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The ability of dopamine (DA) neurons to release other transmitters in addition to DA itself has been increasingly recognized, hence the concept of their multilingual nature. A subset of DA neurons, mainly found in the ventral tegmental area, express VGLUT2, allowing them to package and release glutamate onto striatal spiny projection neurons and cholinergic interneurons. Some dopaminergic axon terminals release GABA. Glutamate release by DA neurons has a developmental role, facilitating axonal growth and survival, and may determine in part the critical contribution of the ventral striatum to psychostimulant-induced behavior. Vesicular glutamate coentry may have synergistic effects on vesicular DA filling. The multilingual transmission of DA neurons across multiple striatal domains and the increasing insight into the role of glutamate cotransmission in the ventral striatum highlight the importance of analyzing DA neuron transmission at the synaptic level., (© 2014 Elsevier B.V. All rights reserved.)

Dopaminergic neurons of the substantia nigra selectively degenerate over the course of Parkinson's disease. These neurons are also the most heavily pigmented cells of the brain, accumulating the dark pigment neuromelanin over a lifetime. The massive presence of neuromelanin in these brain areas has long been suspected as a key factor involved in the selective vulnerability of neurons. The high concentration of neuromelanin in substantia nigra neurons seems to be linked to the presence of considerable amounts of cytosolic dopamine that have not been sequestered into synaptic vesicles. Over the past few years, studies have uncovered a dual nature of neuromelanin. Intraneuronal neuromelanin can be a protective factor, shielding the cells from toxic effects of redox active metals, toxins, and excess of cytosolic catecholamines. In contrast, neuromelanin released by dying neurons can contribute to the activation of neuroglia triggering the neuroinflammation that characterizes Parkinson's disease. This article reviews recent studies on the molecular aspects of neuromelanin of the human substantia nigra.

Vesicular monoamine transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent Km of FFN206 at VMAT2 was found to be 1.16 ± 0.10 μM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z'-factor of 0.7-0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture.

Autism spectrum disorder (ASD) consists of a group of complex developmental disabilities characterized by impaired social interactions, deficits in communication and repetitive behavior. Multiple lines of evidence implicate mitochondrial dysfunction in ASD. In postmortem BA21 temporal cortex, a region that exhibits synaptic pathology in ASD, we found that compared to controls, ASD patients exhibited altered protein levels of mitochondria respiratory chain protein complexes, decreased Complex I and IV activities, decreased mitochondrial antioxidant enzyme SOD2, and greater oxidative DNA damage. Mitochondrial membrane mass was higher in ASD brain, as indicated by higher protein levels of mitochondrial membrane proteins Tom20, Tim23 and porin. No differences were observed in either mitochondrial DNA or levels of the mitochondrial gene transcription factor TFAM or cofactor PGC1α, indicating that a mechanism other than alterations in mitochondrial genome or mitochondrial biogenesis underlies these mitochondrial abnormalities. We further identified higher levels of the mitochondrial fission proteins (Fis1 and Drp1) and decreased levels of the fusion proteins (Mfn1, Mfn2 and Opa1) in ASD patients, indicating altered mitochondrial dynamics in ASD brain. Many of these changes were evident in cortical pyramidal neurons, and were observed in ASD children but were less pronounced or absent in adult patients. Together, these findings provide evidence that mitochondrial function and intracellular redox status are compromised in pyramidal neurons in ASD brain and that mitochondrial dysfunction occurs during early childhood when ASD symptoms appear., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although there have been significant advances, pathogenesis in Parkinson's disease (PD) is still poorly understood. Potential clues about pathogenesis that have not been systematically pursued are suggested by the restricted pattern of neuronal pathology in the disease. In addition to dopaminergic neurons in the substantia nigra pars compacta (SNc), a significant number of other central and peripheral neuronal populations exhibit Lewy pathology (LP), phenotypic dysregulation, or frank degeneration in PD patients. Drawing on this literature, there appears to be a small number of risk factors contributing to vulnerability. These include autonomous activity, broad action potentials, low intrinsic calcium buffering capacity, poorly myelinated long highly branched axons and terminal fields, and use of a catecholamine neurotransmitter, often with the catecholamine-derived neuromelanin pigment. Of these phenotypic traits, only the physiological ones appear to provide a reachable therapeutic target at present., (Copyright © 2013 Movement Disorder Society.)

The Lingo-1 sequence variant has been associated with essential tremor (ET) in several genome-wide association studies. However, the role that Lingo-1 might play in pathogenesis of ET is not understood. Since Lingo-1 protein is a negative regulator of axonal regeneration and neurite outgrowth, it could contribute to Purkinje cell (PC) or basket cell axonal pathology observed in postmortem studies of ET brains. In this study, we used Western blotting and immunohistochemistry to examine Lingo-1 protein in ET vs. control brains. In Western blots, Lingo-1 protein expression level was significantly increased in cerebellar cortex (1.56 ± 0.46 in ET cases vs. 0.99 ± 0.20 in controls, p = 0.002), but was similar in the occipital cortex (p = 1.00) of ET cases vs. controls. Lingo-1 immunohistochemistry in cerebellum revealed that Lingo-1 was enriched in the distal axonal processes of basket cells, which formed a "pinceau" structure around the PC axon initial segment (AIS). We found that some Lingo-1-positive pinceau had abnormally elongated processes, targeting PC axon segments distal to the AIS. In ET cases, the percentage of Lingo-1-positive pinceau that were ≥30 or ≥40 μm in length was increased 2.4- to 4.1-fold, respectively, vs. pinceau seen in control brains (p < 0.0001). Elongated Lingo-1-positive pinceau strongly correlated with number of PC axonal torpedoes and a rating of basket cell axonal pathology. The increased cerebellar Lingo-1 expression and elongated Lingo-1-positive pinceau processes could contribute to the abnormal PC and basket cell axonal pathology and cerebellar dysfunction observed in ET.

We have previously introduced fluorescent false neurotransmitters (FFNs) as optical reporters that enable visualization of individual dopaminergic presynaptic terminals and their activity in the brain. In this context, we examined the fluorescent pyridinium dye 4-(4-dimethylamino)phenyl-1-methylpyridinium (APP+), a fluorescent analogue of the dopaminergic neurotoxin MPP+, in acute mouse brain tissue. APP+ is a substrate for the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT), and as such represented a candidate for the development of new FFN probes. Here we report that APP+ labels cell bodies of catecholaminergic neurons in the midbrain in a DAT- and NET-dependent manner, as well as fine dopaminergic axonal processes in the dorsal striatum. APP+ destaining from presynaptic terminals in the dorsal striatum was also examined under the conditions inducing depolarization and exocytotic neurotransmitter release. Application of KCl led to a small but significant degree of destaining (approximately 15% compared to control), which stands in contrast to a nearly complete destaining of the new generation FFN agent, FFN102. Electrical stimulation of brain slices at 10 Hz afforded no significant change in the APP+ signal. These results indicate that the majority of the APP+ signal in axonal processes originates from labeled organelles including mitochondria, whereas only a minor component of the APP+ signal represents the releasable synaptic vesicular pool. These results also show that APP+ may serve as a useful probe for identifying catecholaminergic innervations in the brain, although it is a poor candidate for the development of FFNs.

To advance understanding of the brain, the ability to measure both nerve cell electrical spiking and chemical neurotransmission with high spatial resolution is required. In comparison to the development of voltage sensors and Ca(2+) indicator dyes over the past several decades, high resolution imaging of neurotransmitter (NT) release at single synapses has not been possible. In this Viewpoint, we discuss two recent developments toward this goal, namely, the design of fluorescent false neurotransmitters (FFNs) and optical neurotransmitter sensors.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. We found LRRK2 to be degraded in lysosomes by chaperone-mediated autophagy (CMA), whereas the most common pathogenic mutant form of LRRK2, G2019S, was poorly degraded by this pathway. In contrast to the behavior of typical CMA substrates, lysosomal binding of both wild-type and several pathogenic mutant LRRK2 proteins was enhanced in the presence of other CMA substrates, which interfered with the organization of the CMA translocation complex, resulting in defective CMA. Cells responded to such LRRK2-mediated CMA compromise by increasing levels of the CMA lysosomal receptor, as seen in neuronal cultures and brains of LRRK2 transgenic mice, induced pluripotent stem cell-derived dopaminergic neurons and brains of Parkinson's disease patients with LRRK2 mutations. This newly described LRRK2 self-perpetuating inhibitory effect on CMA could underlie toxicity in Parkinson's disease by compromising the degradation of α-synuclein, another Parkinson's disease-related protein degraded by this pathway.

We recently introduced fluorescent false neurotransmitters (FFNs) as optical tracers that enable the visualization of neurotransmitter release at individual presynaptic terminals. Here, we describe a pH-responsive FFN probe, FFN102, which as a polar dopamine transporter substrate selectively labels dopamine cell bodies and dendrites in ventral midbrain and dopaminergic synaptic terminals in dorsal striatum. FFN102 exhibits greater fluorescence emission in neutral than acidic environments, and thus affords a means to optically measure evoked release of synaptic vesicle content into the extracellular space. Simultaneously, FFN102 allows the measurement of individual synaptic terminal activity by following fluorescence loss upon stimulation. Thus, FFN102 enables not only the identification of dopamine cells and their processes in brain tissue, but also the optical measurement of functional parameters including dopamine transporter activity and dopamine release at the level of individual synapses. As such, the development of FFN102 demonstrates that, by bringing together organic chemistry and neuroscience, molecular entities can be generated that match the endogenous transmitters in selectivity and distribution, allowing for the study of both the microanatomy and functional plasticity of the normal and diseased nervous system.

An under-appreciated clue about pathogenesis in Parkinson disease (PD) is the distribution of pathology in the early and middle stages of the disease. This pathological 'roadmap' shows that in addition to dopaminergic neurons in the substantia nigra pars compacta (SNc), a significant number of other central and peripheral neuronal populations exhibit Lewy pathology, phenotypic dysregulation or frank degeneration in PD patients. This spatially distributed, at-risk population of neurons shares a number of features, including autonomously generated activity, broad action potentials, low intrinsic calcium buffering capacity and long, poorly myelinated, highly branched axons. Many, and perhaps all, of these traits add to the metabolic burden in these neurons, suggesting that mitochondrial deficits could drive pathogenesis in PD-in agreement with a large segment of the literature. What is less clear is how this neuronal phenotype might shape the susceptibility to proteostatic dysfunction or to the spread of α-synuclein fibrils deposited in the extracellular space. The review explores the literature on these issues and their translational implications.

The mechanistic target of rapamycin (MTOR) has been implicated in regulating synaptic plasticity and neurodegeneration, but MTOR's role in modulating presynaptic function through autophagy is unexplored. We studied presynaptic function in ventral dopamine neurons, a system from which neurotransmitter release can be measured directly by cyclic voltammetry. We generated mutant mice that were specifically deficient for macroautophagy in dopaminergic neurons by deleting the Atg7 gene in cells that express the dopamine uptake transporter. Dopamine axonal profiles in the mutant dorsal striatum were ~one third larger in the mutant mice, released ~50% more stimulus-evoked dopamine release, and exhibited more rapid presynaptic recovery than controls. Rapamycin reduced dopamine neuron axon profile size by ~30% in control mice, but had no effect on macroautophagy deficient axons. Acute rapamycin decreased dopaminergic synaptic vesicle density by ~25% and inhibited evoked dopamine release by ~25% in control mice, but not in the Atg7 deficient mutants. Thus, both basal and induced macroautophagy can provide a brake on presynaptic activity in vivo, perhaps by regulating the turnover of synaptic vesicles, and further regulates terminal volume and the kinetics of transmitter release.

The study of dopamine (DA) quantal size, or the amount of transmitter released per vesicle fusion event, has been enabled by subsecond resolution amperometric recordings. These methods, together with other electrophysiology techniques, novel optical approaches and classical molecular biology and biochemistry methodologies, have advanced our understanding of quantal size regulation in dopaminergic and other catecholaminergic systems. The presynaptic mechanisms that determine DA quantal size regulate two features: the amount of transmitter stored in each vesicle and the fraction of vesicular contents that are released per fusion event. The amount of vesicular DA is dependent on DA synthesis, DA vesicular loading and storage and on DA reuptake from the extracellular space upon exocytosis. The mode of vesicle fusion and the related fusion pore dynamics control the fraction of DA released per fusion event. We will summarize current understanding on the regulation of these steps by endogenous and exogenous factors, including drugs of abuse and DA itself.

mTOR is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and autophagic degradation of cellular components. When triggered by mTOR inactivation, macroautophagy degrades long-lived proteins and organelles via sequestration into autophagic vacuoles. mTOR further regulates synaptic plasticity, and neurodegeneration occurs when macroautophagy is deficient. It is nevertheless unknown whether macroautophagy modulates presynaptic function. We find that the mTOR inhibitor rapamycin induces formation of autophagic vacuoles in prejunctional dopaminergic axons with associated decreased axonal profile volumes, synaptic vesicle numbers, and evoked dopamine release. Evoked dopamine secretion was enhanced and recovery was accelerated in transgenic mice in which macroautophagy deficiency was restricted to dopaminergic neurons; rapamycin failed to decrease evoked dopamine release in the striatum of these mice. Macroautophagy that follows mTOR inhibition in presynaptic terminals, therefore, rapidly alters presynaptic structure and neurotransmission., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Striatal dopamine neurotransmission is critical for normal voluntary movement, affect and cognition. Dysfunctions of its regulation are implicated in a broad range of behaviors and disorders including Parkinson's disease, schizophrenia and drug abuse. Extracellular dopamine levels result from a dynamic equilibrium between release and reuptake by dopaminergic terminals. Both processes are regulated by multiple mechanisms. Here we review data characterizing how dopamine levels are regulated by presynaptic autoreceptors and heteroreceptors, an area intensively investigated due to advances in real time electrochemical detection of extracellular dopamine, i.e., fast-scan cyclic voltammetry and amperometry, and the development of mutant mouse lines with deletions for specific receptors.

Macroautophagy is a cellular mechanism for the clearance of protein aggregates and damaged organelles. Impaired macroautophagy has been observed in neurodegenerative disorders. We investigated the macroautophagy pathway in essential tremor (ET) cases compared to age-matched controls. We analyzed microtubule-associated protein light chain 3-II (LC3-II), S6K, phosphorylated S6K, beclin-1, and mitochondrial membrane proteins levels by Western blot in the post-mortem cerebellum of 10 ET cases and 11 controls. We also performed immunohistochemistry in 12 ET cases and 13 controls to quantify LC3 clustering in Purkinje cells (PCs). LC3-II protein levels were significantly lower in ET cases vs. controls on Western blot (0.84 ± 0.14 vs. 1.00 ± 0.14, p = 0.02), and LC3-II clustering in PCs by immunohistochemistry was significantly lower in ET cases vs. controls (2.03 ± 3.45 vs. 8.80 ± 9.81, p = 0.03). In ET cases, disease duration was inversely correlated with LC3-II protein level (r = -0.64, p = 0.046). We found that mitochondrial membrane proteins were accumulated in ET (TIM23: 1.36 ± 0.11 in ET cases vs. 1.00 ± 0.08 in controls, p = 0.02; TOMM20: 1.63 ± 0.87 in ET cases vs. 1.00 ± 0.14 in controls, p = 0.03). Beclin-1, which is involved in macroautophagy, was strikingly deficient in ET (0.42 ± 0.13 vs. 1.00 ± 0.35, p<0.001). Decreased macroautophagy was observed in the ET cerebellum, and this could be due to a decrease in beclin-1 levels, which subsequently lead to mitochondrial accumulation as a result of autophagic failure. This provides a possible means by which perturbed macroautophagy could contribute to PC pathology in ET.

We present the implementation and application of 4-component relativistic magnetically induced current density using London atomic orbitals for self-consistent field models. We obtain a magnetically balanced basis by a simple scheme where orbitals obtained by imposing restricted kinetic balance are extended by their unrestricted kinetic balance complement. The presented methodology makes it possible to analyze the concept of aromaticity based on the ring current criterion for closed-shell molecules across the periodic table and is independent of the choice of gauge origin. As a first illustration of the methodology we study plots of the magnetically induced current density and its divergence in the series C(5)H(5)E (E = CH, N, P, As, Sb, Bi) at the Kohn-Sham level, as well as integrated ring current susceptibilities, which we compare to previous results (R. Bast et al., Chem. Phys., 2009, 356, 187) obtained using a common gauge origin approach. We find that the current strength decreases monotonically along the series, but that all molecules qualify as aromatic according to the ring current criterion.

A small number of ventral tegmental area dopamine neurons engage in numerous and apparently contradictory functions--how can this be? A clue is provided by Lammel and colleagues in this issue of Neuron: some VTA dopamine neurons display synaptic plasticity in response to cocaine, and others in response to pain, and these populations are distinguished by their axonal projections and Ih., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The fundamental principle that unites addictive drugs appears to be that each enhances synaptic dopamine by means that dissociate it from normal behavioral control, so that they act to reinforce their own acquisition. This occurs via the modulation of synaptic mechanisms that can be involved in learning, including enhanced excitation or disinhibition of dopamine neuron activity, blockade of dopamine reuptake, and altering the state of the presynaptic terminal to enhance evoked over basal transmission. Amphetamines offer an exception to such modulation in that they combine multiple effects to produce nonexocytic stimulation-independent release of neurotransmitter via reverse transport independent from normal presynaptic function. Questions about the molecular actions of addictive drugs, prominently including the actions of alcohol and solvents, remain unresolved, but their ability to co-opt normal presynaptic functions helps to explain why treatment for addiction has been challenging., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A role for dopamine (DA) release in the hallucinations and other positive symptoms associated with schizophrenia has long been inferred from the antipsychotic response to D2 DA receptor antagonists and because the DA releaser amphetamine can be psychotogenic. Recent studies suggest that patients with schizophrenia, including those never exposed to antipsychotic drugs, maintain high presynaptic DA accumulation in the striatum. New laboratory approaches are elucidating mechanisms that control the level of presynaptic DA stores, thus contributing to fundamental understanding of the basic pathophysiologic mechanism in schizophrenia.

Optical imaging is a valuable tool for investigating alterations in membrane turnover and vesicle trafficking. Established techniques can easily be adapted to study the mechanisms of synaptic dysfunction in models of neuropsychiatric disorders and neurodegenerative diseases, such as drug addiction, Parkinsonism, and Huntington's disease. Fluorescent endocytic tracers, including FM1-43, have been used to optically monitor synaptic vesicle fusion and measure synaptic function in various preparations, including chromaffin cells, dissociated cell cultures, and brain slices. In this chapter, we describe a technique that provides a direct measure of pathway-specific exocytosis from glutamatergic corticostriatal terminals.

In Parkinson's disease (PD), there is a progressive loss of neuromelanin (NM)-containing dopamine neurons in substantia nigra (SN) which is associated with microgliosis and presence of extracellular NM. Herein, we have investigated the interplay between microglia and human NM on the degeneration of SN dopaminergic neurons. Although NM particles are phagocytized and degraded by microglia within minutes in vitro, extracellular NM particles induce microglial activation and ensuing production of superoxide, nitric oxide, hydrogen peroxide (H₂O₂), and pro-inflammatory factors. Furthermore, NM produces, in a microglia-depended manner, neurodegeneration in primary ventral midbrain cultures. Neurodegeneration was effectively attenuated with microglia derived from mice deficient in macrophage antigen complex-1, a microglial integrin receptor involved in the initiation of phagocytosis. Neuronal loss was also attenuated with microglia derived from mice deficient in phagocytic oxidase, a subunit of NADPH oxidase, that is responsible for superoxide and H₂O₂ production, or apocynin, an NADPH oxidase inhibitor. In vivo, NM injected into rat SN produces microgliosis and a loss of tyrosine hydroxylase neurons. Thus, these results show that extracellular NM can activate microglia, which in turn may induce dopaminergic neurodegeneration in PD. Our study may have far-reaching implications, both pathogenic and therapeutic.

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M(5) is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M(5) and the AP-3 adaptor complex regulator AGAP1. This interaction was specific with regard to muscarinic receptor (MR) and AGAP subtypes, and mediated the binding of AP-3 to M(5). Interaction with AGAP1 and activity of AP-3 were required for the endocytic recycling of M(5) in neurons, the lack of which resulted in the downregulation of cell surface receptor density after sustained receptor stimulation. The elimination of AP-3 or abrogation of AGAP1-M(5) interaction in vivo decreased the magnitude of presynaptic M(5)-mediated dopamine release potentiation in the striatum. Our study argues for the presence of a previously unknown receptor-recycling pathway that may underlie mechanisms of G-protein-coupled receptor (GPCR) homeostasis. These results also suggest a novel therapeutic target for the treatment of dopaminergic dysfunction.

We introduce pH-responsive fluorescent false neurotransmitters (pH-responsive FFNs) as novel probes that act as vesicular monoamine transporter (VMAT) substrates and ratiometric fluorescent pH sensors. The development of these agents was achieved by systematic molecular design that integrated several structural elements, including the aminoethyl group (VMAT recognition), halogenated hydroxy-coumarin core (ratiometric optical pH sensing in the desired pH range), and N- or C-alkylation (modulation of lipophilicity). Of 14 compounds that were synthesized, the probe Mini202 was selected based on the highest uptake in VMAT2-transfected HEK cells and desirable optical properties. Using Mini202, we measured the pH of catecholamine secretory vesicles in PC-12 cells (pH approximately 5.9) via two-photon fluorescence microscopy. Incubation with methamphetamine led to an increase in vesicular pH (pH approximately 6.4), consistent with a proposed mechanism of action of this psychostimulant, and eventually to redistribution of vesicular content (including Mini202) from vesicles to cytoplasm. Mini202 is sufficiently bright, photostable, and suitable for two-photon microscopy. This probe will enable fundamental neuroscience and neuroendocrine research as well as drug screening efforts.

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, the main process responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntington's disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin protein. However, the precise mechanism behind macroautophagy malfunction in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from humans with HD, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower turnover, functional decay and accumulation inside HD cells.

Dopamine neurons in the ventral tegmental area (VTA) play an important role in the motivational systems underlying drug addiction, and recent work has suggested that they also release the excitatory neurotransmitter glutamate. To assess a physiological role for glutamate corelease, we disrupted the expression of vesicular glutamate transporter 2 selectively in dopamine neurons. The conditional knockout abolishes glutamate release from midbrain dopamine neurons in culture and severely reduces their excitatory synaptic output in mesoaccumbens slices. Baseline motor behavior is not affected, but stimulation of locomotor activity by cocaine is impaired, apparently through a selective reduction of dopamine stores in the projection of VTA neurons to ventral striatum. Glutamate co-entry promotes monoamine storage by increasing the pH gradient that drives vesicular monoamine transport. Remarkably, low concentrations of glutamate acidify synaptic vesicles more slowly but to a greater extent than equimolar Cl(-), indicating a distinct, presynaptic mechanism to regulate quantal size., (Copyright 2010 Elsevier Inc. All rights reserved.)

Alpha-synuclein (alpha-syn) appears to normally regulate neurotransmitter release, possibly via calcium-dependent binding and dissociation from lipid domains on secretory vesicles. The pathogenic effects of alpha-syn leading to Parkinson's disease (PD) appear to result from alternate toxic effects on lipid membrane. A variety of findings indicate that overexpression of wild-type alpha-syn, pathogenic mutations of alpha-syn, and dopamine-modified-alpha-syn promote toxic interaction between alpha-syn oligomers and lipids. These may disrupt transmembrane concentration gradients across secretory vesicles and other organelles and interfere with normal lysosomal or ubiqutin/proteasome mediated protein degradation or mitochondrial function. Additional causes of PD may interfere at other points with normal handling and degradation of alpha-syn, providing a variety of entry points to a converging neurodegenerative path underlying the disease.

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. In order to observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

The basis for selective death of specific neuronal populations in neurodegenerative diseases remains unclear. Parkinson's disease (PD) is a synucleinopathy characterized by a preferential loss of dopaminergic neurons in the substantia nigra (SN), whereas neurons of the ventral tegmental area (VTA) are spared. Using intracellular patch electrochemistry to directly measure cytosolic dopamine (DA(cyt)) in cultured midbrain neurons, we confirm that elevated DA(cyt) and its metabolites are neurotoxic and that genetic and pharmacological interventions that decrease DA(cyt) provide neuroprotection. L-DOPA increased DA(cyt) in SN neurons to levels 2- to 3-fold higher than in VTA neurons, a response dependent on dihydropyridine-sensitive Ca2+ channels, resulting in greater susceptibility of SN neurons to L-DOPA-induced neurotoxicity. DA(cyt) was not altered by alpha-synuclein deletion, although dopaminergic neurons lacking alpha-synuclein were resistant to L-DOPA-induced cell death. Thus, an interaction between Ca2+, DA(cyt), and alpha-synuclein may underlie the susceptibility of SN neurons in PD, suggesting multiple therapeutic targets.

Recessive mutations in parkin are the most common cause of familial early-onset Parkinson's disease (PD). Recent studies suggest that certain parkin mutants may exert dominant toxic effects to cultured cells and such dominant toxicity can lead to progressive dopaminergic (DA) neuron degeneration in Drosophila. To explore whether mutant parkin could exert similar pathogenic effects to mammalian DA neurons in vivo, we developed a BAC (bacterial artificial chromosome) transgenic mouse model expressing a C-terminal truncated human mutant parkin (Parkin-Q311X) in DA neurons driven by a dopamine transporter promoter. Parkin-Q311X mice exhibit multiple late-onset and progressive hypokinetic motor deficits. Stereological analyses reveal that the mutant mice develop age-dependent DA neuron degeneration in substantia nigra accompanied by a significant loss of DA neuron terminals in the striatum. Neurochemical analyses reveal a significant reduction of the striatal dopamine level in mutant mice, which is significantly correlated with their hypokinetic motor deficits. Finally, mutant Parkin-Q311X mice, but not wild-type controls, exhibit age-dependent accumulation of proteinase K-resistant endogenous alpha-synuclein in substantia nigra and colocalized with 3-nitrotyrosine, a marker for oxidative protein damage. Hence, our study provides the first mammalian genetic evidence that dominant toxicity of a parkin mutant is sufficient to elicit age-dependent hypokinetic motor deficits and DA neuron loss in vivo, and uncovers a causal relationship between dominant parkin toxicity and progressive alpha-synuclein accumulation in DA neurons. Our study underscores the need to further explore the putative link between parkin dominant toxicity and PD.

Acute exposure to amphetamines (AMPHs) collapses secretory vesicle pH gradients, which increases cytosolic catecholamine levels while decreasing the quantal size of catecholamine release during fusion events. AMPH and methamphetamine (METH), however, are retained in tissues over long durations. We used optical and electron microscopic probes to measure the effects of long-term METH exposure on secretory vesicle pH, and amperometry and intracellular patch electrochemistry to observe the effects on neurosecretion and cytosolic catecholamines in cultured rat chromaffin cells. In contrast to acute METH effects, exposure to the drug for 6-48 h at 10 microM and higher concentrations produced a concentration-dependent rebound hyperacidification of secretory vesicles. At 5-10 microM levels, prolonged METH increased the quantal size and reinstated exocytotic catecholamine release, although very high (> 100 microM) levels of the drug, while continuing to produce rebound hyperacidification, did not increase quantal size. Secretory vesicle rebound hyperacidification was temperature dependent with optimal response at approximately 37 degrees C, was not blocked by the transcription inhibitor, puromycin, and appears to be a general compensatory response to prolonged exposure with membranophilic weak bases, including AMPHs, methylphenidate, cocaine, and ammonia. Thus, under some conditions of prolonged exposure, AMPHs and other weak bases can enhance, rather than deplete, the vesicular release of catecholamines via a compensatory response resulting in vesicle acidification.

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.

Protein kinase C (PKC) activity mediates multiple neurosecretory processes, but these are poorly understood due in part to the existence of at least 12 PKC isoforms. Using amperometry to record quantal catecholamine release from chromaffin cells, we found that both broad spectrum PKC antagonists and rottlerin, a selective inhibitor of the novel isoforms PKC theta and PKC delta, decreased quantal size and the number of secretory events recorded per stimulus. In contrast, drugs that selectively inhibit the atypical and conventional PKC isoforms had no effect on these parameters. While both PKC theta and delta were expressed in chromaffin cells, mice deficient for PKC theta, but not for PKC delta, exhibited lower quantal size than wild-type and were insensitive to rottlerin. Finally, an inhibitory PKC theta pseudosubstrate produced rottlerin-like responses in wild-type mice, indicating that the lack of rottlerin response in the PKC theta mutants was not the result of a form of compensation. These findings demonstrate neurosecretory regulation by a novel PKC isoform, PKC theta, and should contribute to defining mechanisms of activity-dependent regulation of neurosecretion.

Addiction-associated behaviors such as drug craving and relapse are hypothesized to result from synaptic changes that persist long after withdrawal and are renormalized by drug reinstatement, although such chronic synaptic effects have not been identified. We report that exposure to the dopamine releaser methamphetamine for 10 days elicits a long-lasting (>4 month) depression at corticostriatal terminals that is reversed by methamphetamine readministration. Both methamphetamine-induced chronic presynaptic depression and the drug's selective renormalization in drug-experienced animals are independent of corresponding long-term changes in synaptic dopamine release but are due to alterations in D1 dopamine and cholinergic receptor systems. These mechanisms might provide a synaptic basis that underlies addiction and habit learning and their long-term maintenance.

Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal-lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Altered degradation of alpha-synuclein (alpha-syn) has been implicated in the pathogenesis of Parkinson disease (PD). We have shown that alpha-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of alpha-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic alpha-syn mutations are rare, alpha-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of alpha-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified alpha-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Pacemaking activity in adult substantia nigra (SN) dopamine neurons relies on L-type Ca2+ channels, but a surprising study in Nature by Chan et al. demonstrates that blockade of these channels by dihydropyridines re-establishes the pacemaking driven by sodium and HCN channels found in juvenile SN. This shift protects SN neurons in chemical models of Parkinson's disease (PD), suggesting that elevated intracellular Ca2+ participates in SN cell loss and that dihydropyridines may provide therapy in PD.

Parkinson's disease arises from genetic and possibly neurotoxic causes that produce massive cell death of the neuromelanin-containing dopaminergic neurons of the substantia nigra. Loss of these neurons is essential for the diagnostic parkinsonian features. Although many genetic mutations have been suggested as causes or risk factors for Parkinson's disease, the low penetrance of some mutations and the low disease concordance in relatives suggests that there must be interactions between multiple factors. We suggest that 'multiple hits' that combine toxic stress, for example, from dopamine oxidation or mitochondrial dysfunction, with an inhibition of a neuroprotective response, such as loss of function of parkin or stress-induced autophagic degradation, underlie selective neuronal death. We discuss the properties of substantia nigra dopamine neurons that might make them particular targets of such multiple hits.

Glial cell line-derived neurotrophic factor (GDNF) has shown robust neuroprotective and neuroreparative activities in various animal models of Parkinson's Disease or amyotrophic lateral sclerosis (ALS). The successful use of GDNF as a therapeutic in humans, however, appears to have been hindered by its poor bioavailability to target neurons in the central nervous system (CNS). To improve delivery of exogenous GDNF protein to CNS motor neurons, we employed chemical conjugation techniques to link recombinant human GDNF to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). The predominant species present in the purified conjugate sample, GDNF:TTC, had a molecular weight of approximately 80 kDa as determined by non-reducing SDS-PAGE. Like GDNF, addition of GDNF:TTC to culture media of neuroblastoma cells expressing GFRalpha-1/c-RET produced a dose-dependent increase in cellular phospho-c-RET levels. Treatment of cultured midbrain dopaminergic neurons with either GDNF or the conjugate similarly promoted both DA neuron survival and neurite outgrowth. However, in contrast to mice treated with GDNF by intramuscular injection, mice receiving GDNF:TTC revealed intense GDNF immunostaining associated with spinal cord motor neurons in fixed tissue sections. That GDNF:TTC provided neuroprotection of axotomized motor neurons in neonatal rats further revealed that the conjugate retained its GDNF activity in vivo. These results indicate that TTC can serve as a non-viral vehicle to substantially improve the delivery of functionally active growth factors to motor neurons in the mammalian CNS.

Alpha-synuclein (alpha-syn), a protein implicated in Parkinson's disease pathogenesis, is a presynaptic protein suggested to regulate transmitter release. We explored how alpha-syn overexpression in PC12 and chromaffin cells, which exhibit low endogenous alpha-syn levels relative to neurons, affects catecholamine release. Overexpression of wild-type or A30P mutant alpha-syn in PC12 cell lines inhibited evoked catecholamine release without altering calcium threshold or cooperativity of release. Electron micrographs revealed that vesicular pools were not reduced but that, on the contrary, a marked accumulation of morphologically "docked" vesicles was apparent in the alpha-syn-overexpressing lines. We used amperometric recordings from chromaffin cells derived from mice that overexpress A30P or wild-type (WT) alpha-syn, as well as chromaffin cells from control and alpha-syn null mice, to determine whether the filling of vesicles with the transmitter was altered. The quantal size and shape characteristics of amperometric events were identical for all mouse lines, suggesting that overexpression of WT or mutant alpha-syn did not affect vesicular transmitter accumulation or the kinetics of vesicle fusion. The frequency and number of exocytotic events per stimulus, however, was lower for both WT and A30P alpha-syn-overexpressing cells. The alpha-syn-overexpressing cells exhibited reduced depression of evoked release in response to repeated stimuli, consistent with a smaller population of readily releasable vesicles. We conclude that alpha-syn overexpression inhibits a vesicle "priming" step, after secretory vesicle trafficking to "docking" sites but before calcium-dependent vesicle membrane fusion.

Dysregulation of dopamine homeostasis and elevation of the cytosolic level of the transmitter have been suggested to underlie the vulnerability of catecholaminergic neurons in Parkinson's disease. Because several known mutations in alpha-synuclein or overexpression of the wild-type (WT) protein causes familial forms of Parkinson's disease, we investigated possible links between alpha-synuclein pathogenesis and dopamine homeostasis. Chromaffin cells isolated from transgenic mice that overexpress A30P alpha-synuclein displayed significantly increased cytosolic catecholamine levels as measured by intracellular patch electrochemistry, whereas cells overexpressing the WT protein and those from knock-out animals were not different from controls. Likewise, catechol concentrations were higher in L-DOPA-treated PC12 cells overexpressing A30P or A53T compared with those expressing WT alpha-synuclein, although the ability of cells to maintain a low cytosolic dopamine level after L-DOPA challenge was markedly inhibited by either protein. We also found that incubation with low-micromolar concentrations of WT, A30P, or A53T alpha-synuclein inhibited ATP-dependent maintenance of pH gradients in isolated chromaffin vesicles and that the WT protein was significantly less potent in inducing the proton leakage. In summary, we demonstrate that overexpression of different types of alpha-synuclein disrupts vesicular pH and leads to a marked increase in the levels of cytosolic catechol species, an effect that may in turn trigger cellular oxyradical damage. Although multiple molecular mechanisms may be responsible for the perturbation of cytosolic catecholamine homeostasis, this study provides critical evidence about how alpha-synuclein might exert its cytotoxicity and selectively damage catecholaminergic cells.

Microdeletions of 22q11.2 represent one of the highest known genetic risk factors for schizophrenia. It is likely that more than one gene contributes to the marked risk associated with this locus. Two of the candidate risk genes encode the enzymes proline dehydrogenase (PRODH) and catechol-O-methyltransferase (COMT), which modulate the levels of a putative neuromodulator (L-proline) and the neurotransmitter dopamine, respectively. Mice that model the state of PRODH deficiency observed in humans with schizophrenia show increased neurotransmitter release at glutamatergic synapses as well as deficits in associative learning and response to psychomimetic drugs. Transcriptional profiling and pharmacological manipulations identified a transcriptional and behavioral interaction between the Prodh and Comt genes that is likely to represent a homeostatic response to enhanced dopaminergic signaling in the frontal cortex. This interaction modulates a number of schizophrenia-related phenotypes, providing a framework for understanding the high disease risk associated with this locus, the expression of the phenotype, or both.

Amperometry is widely used to study exocytosis of neurotransmitters and hormones in various cell types. Analysis of the shape of the amperometric spikes that originate from the oxidation of monoamine molecules released during the fusion of individual secretory vesicles provides information about molecular steps involved in stimulation-dependent transmitter release. Here we present an overview of the methodology of amperometric signal processing, including (i) amperometric signal acquisition and filtering, (ii) detection of exocytotic events and determining spike shape characteristics, and (iii) data manipulation and statistical analysis. The purpose of this review is to provide practical guidelines for performing amperometric recordings of exocytotic activity and interpreting the results based on shape characteristics of individual release events.

Septin 5, a parkin substrate, is a vesicle- and membrane-associated protein that plays a significant role in inhibiting exocytosis. The regulatory function of Septin 5 in dopaminergic (DAergic) neurons of substantia nigra (SN), maintained at relatively low levels, has not yet been delineated. As loss of function mutations of parkin are the principal cause of a familial Parkinson's disease, a prevailing hypothesis is that the loss of parkin activity results in accumulation of Septin 5 which confers neuron-specific toxicity in SN-DAergic neurons. In vitro and in vivo models were used to support this hypothesis. In our well-characterized DAergic SN4741 cell model, acute accumulation of elevated levels of Septin 5, but not synphilin-1 (another parkin substrate), resulted in cytotoxic cell death that was markedly reduced by parkin co-transfection. A transgenic mouse model expressing a dominant negative parkin mutant accumulated moderate levels of Septin 5 in SN-DAergic neurons. These mice acquired a progressive l-DOPA responsive motor dysfunction that developed despite a 25% higher than normal level of striatal dopamine (DA) and no apparent loss of DAergic neurons. The phenotype of this animal, increased striatal dopamine and reduced motor function, was similar to that observed in parkin knockout animals, suggesting a common DAergic alteration. These data suggest that a threshold level of Septin 5 accumulation is required for DAergic cell loss and that l-DOPA-responsive motor deficits can occur even in the presence of elevated DA.

Using a cell fusion assay, we show here that in addition to complete fusion SNAREs also promote hemifusion as an alternative outcome. Approximately 65% of events resulted in full fusion, and the remaining 35% in hemifusion; of those, approximately two thirds were permanent and approximately one third were reversible. We predict that this relatively close balance among outcomes could be tipped by binding of regulatory proteins to the SNAREs, allowing for dynamic physiological regulation between full fusion and reversible kiss-and-run-like events.

Neurotransmitter transporters have long been known to recognize related compounds as substrates, resulting in the accumulation and release of so-called "false transmitters." In this issue of Neuron, Zhou et al. show that when serotonin levels are elevated by inhibition of either serotonin reuptake or of monoamine oxidase, dopamine neurons accumulate serotonin. The results suggest that release of serotonin by dopamine neurons may contribute to the effects of multiple major classes of antidepressants.

Amphetamine and substituted amphetamines, including methamphetamine, methylphenidate (Ritalin), methylenedioxymethamphetamine (ecstasy), and the herbs khat and ephedra, encompass the only widely administered class of drugs that predominantly release neurotransmitter, in this case principally catecholamines, by a non-exocytic mechanism. These drugs play important medicinal and social roles in many cultures, exert profound effects on mental function and behavior, and can produce neurodegeneration and addiction. Numerous questions remain regarding the unusual molecular mechanisms by which these compounds induce catecholamine release. We review current issues on the two apparent primary mechanisms--the redistribution of catecholamines from synaptic vesicles to the cytosol, and induction of reverse transport of transmitter through plasma membrane uptake carriers--and on additional drug effects that affect extracellular catecholamine levels, including uptake inhibition, effects on exocytosis, neurotransmitter synthesis, and metabolism.