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1 Introduction

It is important to recognize that neurodegenerative disorders involve not only loss of neurons, but significant dysfunction in select populations of remaining neurons. This impaired function of remaining but injured neurons manifests in disruption of neurotransmission. This chapter describes the impact of oxidative stress and deficits in cellular energy and mitochondrial function on neurotransmission, particularly in the context of neurodegenerative disease. A functional window into neural injury enables elucidation of early injuries. The roles these deficits are thought to play in the early aetiology of several neurodegenerative diseases are highlighted. The theory underlying pertinent techniques used to study exocytosis and neurotransmission and the application of these techniques in the study of neurodegenerative disease are also described.

2 Synaptic Transmission, Oxidative Stress and Mitochondrial Dysfunction

2.1 Gene Responses to Normal Ageing in the Brain

While the factors underlying cognitive decline in both normal ageing and neurodegenerative diseases are not clearly understood, reductions in the amount and efficiency of synaptic transmission are obvious focal points. Synaptic transmission decreases during normal ageing [1, 2] and in the early stages of disease pathology in animal models of some neurodegenerative diseases [3–8]. A postmortem analysis of gene expression in the frontal cortex of human subjects aged 26–106 revealed that the expression of genes involved in synaptic plasticity, vesicular transport and mitochondrial function decrease after 40 years of age [9]. However, genes involved in the stress response, antioxidation and DNA repair are induced after 40 years of age [9]. DNA damage is also markedly increased in the promoters of genes that show reduced expression in the aged cortex and these same genes are less capable of undergoing DNA repair [9].

2.2 Oxidative Injury in Normal Ageing with Impact on Synaptic Transmission

Oxidative damage is a likely contributing factor to the cognitive decline that accompanies ageing. Mitochondrial dysfunction, amounts of reactive oxygen species (ROS) and prolonged periods of oxidative stress increase with age (reviewed in [10]). Sources of ROS, in the form of O2 •  –, and hence ultimately oxidative stress, include mitochondria, NADPH oxidase, xanthine oxidase and P450 oxidase. Throughout this text, the term ROS will be used in reference to hydrogen peroxide (H2O2). The usual reference is to the superoxide anion (O2 • ), which is produced during the reduction of O2 to water in the mitochondrial electron transport chain. As O2 •  − is spontaneously or enzymatically converted to H2O2 at rapid rates, the term ROS usually refers ultimately to H2O2, as it is far more long-lived than O2 •  − within cells.

Increasing amounts of ROS have been attributed to impairing long-term potentiation (LTP), an increase in the strength of a neuronal synapse, in hippocampal area CA1 in an age-related manner as determined by measuring excitatory postsynaptic potentials (EPSPs) from hippocampal slices [1, 11]. Hippocampal slices from older rats produce more H2O2 than younger rats (reviewed in [12]) and age is associated with decreases in the hippocampal concentration of vitamins C and E and increased activity of superoxide dismutase [13]. The concentrations of ROS applied to these slices have differential effects on LTP. For example, low concentrations (1 μM) of H2O2 potentiate LTP in hippocampus of young rats by up to two-fold by affecting internal calcium (Ca2+) stores [14]. However, high concentrations (20 μM or above) of H2O2 impair synaptic transmission and LTP via calcineurin-dependent mechanisms [14]. These findings indicate that optimal amounts of ROS are required for the highest levels of LTP to occur and too few or too many ROS are detrimental to synaptic transmission. The requirement for an optimal amount of ROS is also evident in other aspects of normal cellular function, such as the regulation of neuronal excitability via redox-sensitive ion channels [15–18], synaptic plasticity [12], gene transcription [19], multiple signal transduction pathways [19] and the activity of enzymes controlling protein phosphorylation [20].

2.3 Redox Regulation of Exocytosis

The synaptic fusion machinery can be directly and acutely regulated by redox state. This was illustrated in frog and mouse motor nerve endings, where evoked and spontaneous quantal release was reduced by physiological levels of ROS [21]. Antioxidants also increased the quantal level of release, consistent with the tonic inhibition of exocytosis by endogenous levels of ROS [21]. The use of a Ca2+ ionophore subsequently revealed that this effect of ROS was due to action directly on the fusion machinery [21]. Subsequent investigations established that some SNARE proteins are sensitive to oxidative stress, SNAP-25 being the most sensitive of those studied [21]. Specific cysteine residues in SNAP-25 are required for SNARE disassembly and exocytosis but not for membrane targeting [22]. As cysteine residues are commonly affected by redox state, crucial alterations in SNAP-25 structure may underlie the lack of SNARE complex assembly and reduced exocytosis seen in motor nerve endings during conditions of oxidative stress. Interestingly, the expression of some exocytotic proteins, including SNARE proteins, is altered in disease-relevant brain areas in neurodegenerative diseases such as AD and HD [23, 24]. Therefore, the redox modulation of SNARE complex formation is a mechanism by which mitochondrial dysfunction occurring early in some neurodegenerative diseases might reduce synaptic activity.

2.4 Energy-Dependence of Exocytosis

Certain steps in the exocytotic pathway are also regulated by ATP in the cell. The two major ATP-regulated steps in exocytosis have been identified as a reversible ATP-dependent priming of docked granules, which is followed by a second, Ca2+-dependent step involving vesicle fusion and the release of vesicle contents [25, 26]. The ATP-dependent step comprises both vesicle recruitment to the plasma membrane and vesicle priming [27, 28]. The role of ATP here is to enable the phosphorylation of phosphatidylinositol groups via phosphatidylinositol kinases localized to both vesicle and plasma membranes. These kinases include phosphatidylinositol-4-phosphate 5-kinase [29], phosphatidylinositol 4-kinase [30] and phosphatidylinositol 3-kinase C2α [31]. While there is some association between neurodegenerative diseases and the effects of ATP on exocytosis, most of this evidence is indirect. An example of this is the overexpression of the PD-associated protein α-synuclein inhibiting a vesicle-priming step prior to Ca2+-dependent vesicle fusion but after secretory vesicle trafficking to docking sites [32]. While it is unknown how this inhibition occurs, the step at which it takes place is also where ATP-dependent priming occurs.

Two recent independent studies on Drosophila mutants with synapses containing few or no mitochondria support the hypothesis that proper synaptic mitochondrial ATP production is vital for normal synaptic transmission and that mitochondria play a specific role in regulating synaptic strength [33, 34]. Flies with loss-of-function mutations in dynamin-related protein (drp), which is implicated in the fission of the outer mitochondrial membrane, display almost no synaptic mitochondria. These animals sometimes develop into adult flies and the mitochondria in cell bodies remain functional, allowing the role of synaptic mitochondria in neurotransmission to be assessed. In the neuromuscular junction of these mutants, resting Ca2+ levels are slightly increased and basal synaptic properties are hardly altered. However, during intense stimulation, normal neurotransmission cannot be maintained [34]. This is not due to alterations in the kinetics of vesicle fusion or fission, but rather stems from a reduced ability to mobilize the reserve pool of vesicles, an effect that is partially rescued by exogenous ATP [34].

Drosophila mutants of Miro (dmiro), a mitochondrial GTPase with an essential role in mitochondrial trafficking, exhibit defects in locomotion and die prematurely [33]. Neuronal and muscular mitochondria in dmiro mutants are not transported into axons and dendrites but accumulate in neuronal somata. Similarly to drp mutants, dmiro mutants only display changes in neurotransmission during prolonged stimulation [33]. Given the considerable reduction in synaptic mitochondria, it might be presumed that amounts of ROS would be considerably reduced in these mutants. As previously stated, a significant reduction in ROS can be detrimental to multiple facets of neuronal function. Unfortunately, neither of the studies mentioned [33, 34] measured synaptic ROS levels in these mutant flies, so assessment of the separate effect of mitochondrial ATP and ROS on neurotransmission in this setting cannot be made. However, these studies highlight the requirement for synaptic mitochondrial ATP during periods of intense activity for the recruitment and transport of vesicles from the reserve vesicle pool (Fig. 1).

Fig. 1
figure 4_1_150867_1_En

Mitochondrial and vesicular trafficking, mitochondrial function, exocytosis and LTP are altered in various neurodegenerative diseases. (A) Axonal mitochondria and vesicle trafficking are regulated by mutant Htt in ALS patients and in Drosophila mutants of dmiro and drp in which a lack of mitochondrial ATP results in reduced recruitment and transport of vesicles from the reserve vesicle pool during prolonged stimulation. Exocytosis and/or LTP are reduced by the factors indicated in the white box (B) Disease-associated proteins which either reduce mitochondrial ATP production or increase mitochondrial ROS levels. (C) Simple schematic illustrating the points at which ATP and ROS regulate exocytosis. ATP is needed for trafficking of vesicles towards the plasma membrane and then for ‘priming of vesicles for release. ROS inhibit the formation of SNARE protein complexes and therefore inhibit the final steps in vesicle fusion. Increased ROS may either reduce the amount of SNARE proteins available and may also reduce its function

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3 Neurodegenerative Disorders

There is extensive evidence supporting a role for mitochondrial dysfunction and oxidative stress in the pathogenesis of multiple neurodegenerative disorders and in animal models of these diseases. Mitochondria are significantly more likely to be localized at synaptic sites [35] and the speed of mitochondrial motility is greatest in younger, developing neurons [36]. Mitochondria are increasingly localized to dendritic spines during early developmental stages or following repeated stimulation [37]. This mitochondrial localization is necessary for the normal development and morphological plasticity of dendritic spines [37]. The following section describes studies that provide evidence for roles of mitochondrial dysfunction, oxidative stress and cell signalling in the early aetiologies of some neurodegenerative diseases.

3.1 Alzheimer s Disease

In human Alzheimer s disease (AD) patients, signs of oxidative damage are observed earlier in the disease course than the characteristic plaques [38]. Similar observations were also made in transgenic amyloid precursor protein (APP) mice, in which the upregulation of genes involved in mitochondrial metabolism and apoptosis precede Aβ deposition [39, 40]. Oxidative stress may be a causative agent of AD as it induces the intracellular accumulation of Aβ. Secretion of neuronal Aβ into the extracellular space is positively correlated with synaptic activity [41–43] and intraneuronal Aβ accumulation is linked with early electrophysiological, synaptic and pathological abnormalities (reviewed in [44]). Intracellular Aβ accumulation is reduced by antibodies against Aβ in transgenic mice [45] and this reduction currently provides the best correlate for improving cognitive function [46]. It is possible that Aβ accumulation caused by oxidative stress is due to reduced exocytosis from neurons. Various mouse models of AD manifest synaptic dysfunction, including LTP deficits, well before classical pathologies such as neurodegeneration and plaque and tangle formation are evident [3, 6, 7]. Similar results are also seen in nonmammalian models of AD, such as in Drosophila, where the deletion or overexpression of the Drosophila APP orthologue, App-like gene, results in reduced axonal transport of synaptic vesicles [47].

3.2 Parkinson s Disease

Mitochondrial dysfunction and vesicular abnormalities have also been implicated in Parkinson s disease (PD). Several genes associated with PD have roles in mitochondrial function, such as Parkin and PINK-1. The inhibition of complex I of the mitochondrial electron transport chain has been shown to cause widespread neuronal damage [48]. This damage includes PD-like pathology, such as nigrostriatal dopaminergic degeneration and α-synuclein-positive cytoplasmic inclusions [48]. The mechanisms underlying these changes are thought to involve oxidative stress [49].

The role of α-synuclein, a presynaptic protein, is thought to be central in PD pathogenesis. However it remains unclear exactly what role α-synuclein plays in the development of PD. In transgenic mice overexpressing α-synuclein, mitochondrial function is impaired, oxidative stress is increased and nigral degeneration, induced by inhibitors of mitochondrial complex I, is enhanced [50]. α-synuclein at normal expression levels appears to be neuroprotective in nondopaminergic human cortical neurons but induces apoptosis when overexpressed in the dopaminergic neurons affected in PD [51]. Ablation or reduction of α-synuclein expression reduces transmitter release, LTP and the size of vesicle pools [32, 52, 53]. Mice overexpressing a dominant-negative mutation of α-synuclein exhibit decreased dopamine release in response to prolonged stimulation [54, 55]. However, while the mutations in α-synuclein that cause genetic forms of PD are clinically of interest, no clear evidence has been published to clearly illustrate that cellular levels of free α-synuclein are increased or decreased in PD brains and thus it is difficult to assess exactly what role α-synuclein plays in the pathogenesis of sporadic forms of PD.

Parkin, a PD-related ubiquitin ligase, is thought to be protective of mitochondria. In Parkin-null mice, oxidative stress and mitochondrial dysfunction are evident [56]. Parkin also seems to be susceptible to oxidative stress, as S-nitrosylation of Parkin reduces its ubiquitin-ligase activity as well as its protective function [57]. Similar effects are also seen in vivo in a mouse model of PD and in the brains of patients with PD [57]. PD mouse models exhibit various alterations in dopaminergic neuronal function prior to the degeneration of dopaminergic brain areas [55, 58, 59].

The role of PINK-1 has also been studied using PINK-1 null mice. The numbers of dopaminergic neurons and dopamine content in the striatum are unaltered in PINK-1 null mice [60]. However, evoked dopamine release from striatal slices is reduced, as is LTP in striatal medium spiny neurons, the main target of dopaminergic neurons [60]. These results, and those mentioned previously, indicate that altered dopaminergic exocytosis may be a pathogenic precursor to nigrostriatal degeneration in PD.

3.3 Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS), commonly known as motor neuron disease, is another neurodegenerative disease with a mitochondrial and oxidative stress component. Mutations in the Cu/Zn-superoxide dismutase gene account for approximately 20% of familial ALS (FALS) cases. Spinal cords and brains obtained from mice that overexpress a SOD1 mutation (G93A) found in some FALS patients display defects in mitochondrial respiration, electron transfer chain activity and ATP synthesis. Signs of oxidative damage to mitochondrial proteins and lipids, indicative of oxidative stress at the time of disease onset, are also evident [61]. Changes in mitochondrial function are seen before this stage though, as mitochondria in the spinal cord and brain display reduced Ca2+ loading ability in these mice [62]. Mitochondria in the proximal axons of anterior horn neurons also appear to be aberrantly localized to the axon hillock in sporadic ALS patients and mouse models of ALS [63, 64]. This was observed in transgenic mice expressing the G93A mutant human SOD1 during presymptomatic stages, indicating that alterations in mitochondrial localization may occur prior to the onset of ALS [64]. Early changes in synaptic transmission are also seen in mouse models of ALS prior to the onset of neurodegeneration [65, 66].

3.4 Huntington s Disease

Huntington s Disease (HD) is also thought to have a mitochondrial component. In neurons of huntingtin knock-in mice, ATP production and mitochondrial respiration are decreased [67], as are the activities of complexes I and III of the electron transport chain in human HD brain samples [68]. These effects may be caused by direct interaction of the huntingtin gene with mitochondria. Mutant huntingtin is localized to neuronal mitochondrial membranes and incubation of mutant huntingtin with normal mitochondria results in depolarization of the mitochondrial membrane at lower Ca2+ loads. This defect has also been observed in mitochondria obtained from HD patients and mice that transgenically overexpress mutant huntingtin [69].

Huntingtin is involved in axonal trafficking in mammals and mutant huntingtin impairs the in vitro and in vivo trafficking of vesicles and mitochondria in mammalian neurons [70]. Importantly, these changes are seen prior to the development of observed neurological abnormalities [70], implying a possible causative role for this altered mitochondrial localization in axons in HD. Huntingtin also interacts with the transcription factor p53 [71], which is known to regulate genes that are responsive to oxidative stress as well as genes which regulate mitochondrial function. In both Drosophila and mouse models of HD, p53 has been found to link nuclear and mitochondrial pathologies characteristic of HD [72]. Altered synaptic plasticity in the R6/2 mouse model of HD occurs prior to the more overt disease phenotypes [5], indicating that synaptic pathology also occurs early in this disease.

4 Methods Used to Study Exocytosis

The following sections attempt to provide a detailed explanation of some of the more common techniques used in the study of exocytosis and synaptic transmission. These methods are relevant to many of the studies mentioned in this chapter. Along with an explanation of the methodology itself, we have also attempted to profile the pros and cons associated with the use of individual techniques and the types of preparations they can be applied to.

4.1 Carbon Fibre Amperometry and Cyclic Voltammetry

Carbon fibre amperometry is a technique used to detect exocytosis in cells that secrete oxidizable molecules such as adrenaline, noradrenaline, dopamine and serotonin. It involves placing a carbon fibre electrode on a single cell in culture or in a brain slice preparation. The cell is then stimulated to trigger exocytosis, releasing vesicle contents that are then oxidized upon contact with the charged carbon fibre electrode. This oxidation results in a flow of current which can be recorded as an amperometric spike (Fig. 2A).

Fig. 2
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Example recordings of carbon fibre amperometry and whole cell membrane capacitance measurements from mouse chromaffin cells. (A) Carbon fibre amperometry recording of exocytosis. The line beneath the trace indicates where the cell has been stimulated. Current spikes indicate the release of catecholamines from an individual vesicle. (B) Membrane capacitance measurement of exocytosis in which exocytosis is elicited by a repeated set of voltage pulses from –70 to 0 mV. The membrane capacitance is measured in between these pulses and increases accordingly. Scale bar in (B) represents 50 pA and 10 s in (A) and 200 fF and 1 s in (B)

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Amperometry is useful as it is noninvasive and does not alter the intracellular environment. It is possible to visualize single exocytotic events providing the stimulus to the cell is not too great and does not cause overlap of individual amperometric spikes. This allows the shape of individual spikes to be studied, providing insight into the kinetic parameters of individual fusion events. Such parameters include the amount of transmitter released from a single vesicle, the speed of fusion pore opening and closing and the kinetics associated with the formation of the transient fusion pore.

Studies of exocytosis using amperometry have shown that, in addition to the classical model of full collapse fusion, where the vesicle membrane fully collapses into the plasma membrane, transient fusion events occur that also result in secretion [73–75]. Amperometry has also provided insight into the kinetics of vesicle fusion and the mechanisms behind it in a diverse array of cell types, including chromaffin cells, dopaminergic neurons, PC12 cells and pancreatic β-cells [76–80]. Whilst pancreatic β-cells do not secrete endogenous compounds which can be oxidized, they can be loaded with a ‘false oxidizable transmitter such as dopamine. However, in our experience, this loading of false transmitter works poorly and is highly unreliable.

Amperometry is limited to cells that secrete oxidizable molecules and by the geometry of the electrode and the cell, as exocytotic events will only be detected when they occur within the proximity of the electrode. This necessitates the use of large sample sizes to reduce the random effects of signal diffusion from exocytotic events more distant from the electrode and also due to the Gaussian distribution of vesicle size within a cell. Amperometry has been utilized in the studies described in this section to assess the effects of PINK1 inactivation on catecholamine release from dissociated adrenal chromaffin cells and the release of dopamine from acute striatal slices obtained from PINK1 −/− mice [60]. Amperometry has also been used to study secretion from dissociated chromaffin cells isolated from mice overexpressing α-synuclein [32].

It is not possible to determine the identity of the molecules secreted from cells using carbon fibre amperometry. This information is discernable using a similar technique called cyclic voltammetry, which involves constantly scanning the electrical potential of the electrode through a range of voltages [80]. The identity of the molecule is determined according to the potential at which it is oxidized or reduced [80]. Cyclic voltammerty has been used to study dopamine release from neurons in vivo, in mice where the human α-synuclein gene has been overexpressed, mutated or knocked out, and in vitro, such as in brain slices obtained from DJ-1 null mice [54, 55, 58].

4.2 Patch Clamp Capacitance and Postsynaptic Potential Measurement

Measurement of cell membrane capacitance via whole cell patch clamp provides another means to study exocytosis (Fig. 2B) [81]. Vesicles that fuse with the cell membrane cause an increase in membrane area, which can be detected as an increase in membrane capacitance (a capacitor being any material that separates two charged environments). Carrying out capacitance measurements in ‘Cell-Attached mode allows single exocytotic events to be resolved on a millisecond timescale. Unlike amperometry and voltammetry, patch clamping is not limited to cells that secrete oxidizable molecules. However, patch clamping is invasive and can disturb the intracellular environment. Most importantly, it is not applicable to the direct study of exocytosis in most types of neurons as the synapse is too small to place a microelectrode onto and obtain a seal. Recordings can be made at the neuronal soma but this places the microelectrode too electrically distant from the site of synaptic release, precluding direct measurement of presynaptic exocytosis. Such recordings are limited to neurons with relatively large synapses, such as the Calyx of Held and ribbon synapses in the inner ear and retina. Studies using these cells have provided a greater understanding of the mechanisms underlying neuronal exocytosis.

Another application of patch clamping is in the study of postsynaptic currents in neurons. Such experiments involve patch clamping a neuron in culture or in a brain slice preparation then applying electrical or pharmacological stimuli to neurons that synapse onto the patched neuron. Measuring the postsynaptic response elicited by the stimulated neuron provides insights into the exocytotic activity elicited in the presynaptic neuron [60]. This method is advantageous as it allows recordings to be obtained in a system that is relatively more representative of the in vivo environment. However, recording from postsynaptic neurons following a stimulus applied presynaptically in order to gauge presynaptic levels of exocytosis assumes that the applied experimental conditions have only affected presynaptic vesicle release and not the postsynaptic response. This is a popular assumption but one in which extreme care should be taken when making.

Insights into synaptic dysfunction and memory deficits in murine models of neurodegenerative diseases have been obtained by measuring field excitatory postsynaptic potentials. These measurements are obtained using field electrodes to detect action potentials from a population of neurons in hippocampal slices in response to electrical stimulation. Such measurements give insights into synaptic plasticity, learning and memory by providing a comparison of stimulus intensity and the speed and strength of the resulting postsynaptic potential [3, 5–7, 52, 65, 66].

4.3 Fluorescent Dyes and Live Cell Imaging

Fluorescent styryl compound dyes, commonly known as FM dyes (such as FM1-43], reversibly stain membranes and can be used to give a fluorescent readout of exocytosis and endocytosis. These compounds emit a low fluorescence in aqueous solutions which increases upon membrane binding. Stimulating cells in the presence of FM dyes in the extracellular solution results in dye becoming trapped in recycling vesicles, which can be visualized as punctate fluorescence within cells. Upon exocytosis, the trapped dye is released and the rate and the amount of decrease are indicative of the kinetics of exocytosis and the size of the vesicle recycling pool, respectively. Studies using FM dyes have yielded information on the rate and regulation of vesicle exocytosis and recycling in numerous cell types, such as frog neuromuscular junctions, hippocampal neurons and chromaffin cells [82–84].

pHluorins are another set of fluorescent molecules used to study exocytosis. These molecules consist of a mutant form of green fluorescent protein that displays pH-dependent fluorescence [85]. One such molecule is synapto-pHlourin consisting of a pHlourin molecule fused to VAMP2, a synaptic vesicle protein involved in SNARE formation and vesicle exocytosis [85]. Synapto-pHluorin fluorescence is quenched in the relatively low pH of the vesicle lumen (approximately 5.6]. During exocytosis, exposure of the vesicle lumen to the extracellular environment increases the luminal pH to approximately 7.4, resulting in increased fluorescence of the synapto-pHluorin and a visual indication of exocytosis [85]. Synapto-pHluorins have been used to study exocytosis and vesicle recycling in real time, in both astrocytes and neurons [85–88].

Fluorescent dyes are also available that specifically bind to Ca2+ within cells. These are important tools in the study of exocytosis as Ca2+ entry into the cell is the major physiological trigger of vesicle exocytosis and recycling. These dyes, such as fura-2, are fluorophores linked to calcium chelators. When the calcium chelator binds to calcium in a cell, the absorption spectrum of the fluorophore is altered, leading to fluorescence and a visual readout of changes in intracellular Ca2+ levels. This technique is useful in experiments using groups of cells, as the excitation of all cells present can be assessed simultaneously. However, it is not sensitive enough to detect small influxes of calcium that could still be sufficient to stimulate exocytosis [89, 90]. It is also technically difficult, but not impossible, to measure changes in intracellular Ca2+ levels in structurally limited areas such as synaptic boutons. Thus, imaging synaptic Ca2+ changes while simultaneously measuring neuronal exocytosis is not frequently performed.

5 Conclusion

Identifying the steps which instigate human neurodegenerative diseases is extremely complex and continues to elude researchers within the field. Studies described in this chapter provide evidence for an early disease course involving mitochondrial dysfunction leading to aberrant ROS and ATP levels. These abnormalities then lead to reduced synaptic activity and altered neuronal communication, underlying cognitive deficits and sometimes motor dysfunction, and possibly neuronal death. Whilst there is evidence supporting this hypothesis, it would be imprudent to believe it is the only compelling hypothesis on this topic or that it might be responsible for all facets of these diseases. The use of animal models for these diseases will likely prove to be invaluable in identifying the precise sequence of events leading to neurodegeneration and the subsequent verification of these findings in human samples. Proper use of techniques which enable neuronal exocytosis and vesicle recycling and trafficking to be measured will also play a vital part in increasing our understanding of the pathogenesis of such diseases. An improved understanding of the pathogenesis of neurodegenerative diseases will hopefully lead to treatments that are increasingly effective and disease specific.