Biomedical subjects

Robert H Edwards

Publications and source records attributed to Robert H Edwards.

At least 37 records · Page 2Linked to original sources

PubMed2004

Vesicular glutamate transporter 3 expression identifies glutamatergic amacrine cells in the rodent retina.

Synaptic transmission from glutamatergic neurons requires vesicular glutamate transporters (VGLUTs) to concentrate cytosolic glutamate in synaptic vesicles. In retina, glutamatergic photoreceptors and bipolar cells exclusively express the VGLUT1 isoform, whereas ganglion cells express VGLUT2. Surprisingly, the recently identified VGLUT3 isoform was found in presumed amacrine cells, generally considered to be inhibitory interneurons. To investigate the synaptic machinery and conceivable secondary neurotransmitter composition of VGLUT3 cells, and to determine a potential functional role, we further investigated these putative glutamatergic amacrine cells in adult and developing rodent retina. Reverse transcriptase-PCR substantiated VGLUT3 expression in mouse retina. VGLUT3 cells did not immunostain for ganglion or bipolar cell markers, providing evidence that they are amacrine cells. VGLUT3 colocalized with synaptic vesicle markers, and electron microscopy showed that VGLUT3 immunostained synaptic vesicles. VGLUT3 cells were not immunoreactive for amacrine cell markers gamma-aminobutyric acid, choline acetyltransferase, calretinin, or tyrosine hydroxylase, although they immunostain for glycine. VGLUT3 processes made synaptic contact with ganglion cell dendrites, suggesting input onto these cells. VGLUT3 immunostaining was closely associated with the metabotropic glutamate receptor 4, which is consistent with glutamatergic synaptic exocytosis by these cells. In the maturing mouse retina, Western blots showed VGLUT3 expression at postnatal day 7/8 (P7/8). VGLUT3 immunostaining in retinal sections was first observed at P8, achieving an adult pattern at P12. Thus, VGLUT3 function commences around the same time as VGLUT1-mediated glutamatergic transmission from bipolar cells. Furthermore, a subset of VGLUT3 cells expressed the circadian clock gene period 1, implicating VGLUT3 cells as part of the light-entrainable retina-based circadian system.

Amacrine Cells↗

PubMed2004

Lipid rafts mediate the synaptic localization of alpha-synuclein.

Alpha-synuclein contributes to the pathogenesis of Parkinson's disease (PD), but its precise role in the disorder and its normal function remain poorly understood. Consistent with a presumed role in neurotransmitter release and its prominent deposition in the dystrophic neurites of PD, alpha-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, alpha-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of alpha-synuclein with cell membranes, we find that alpha-synuclein binds saturably and with high affinity to characteristic intracellular structures that double label for components of lipid rafts. Biochemical analysis demonstrates the interaction of alpha-synuclein with detergent-resistant membranes and reveals a shift in electrophoretic mobility of the raft-associated protein. In addition, the A30P mutation associated with PD disrupts the interaction of alpha-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute alpha-synuclein away from synapses, indicating an important role for raft association in the normal function of alpha-synuclein and its role in the pathogenesis of PD.

Amino Acid Substitution↗

PubMed2004

Endocannabinoid-independent retrograde signaling at inhibitory synapses in layer 2/3 of neocortex: involvement of vesicular glutamate transporter 3.

Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex. Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laser-scanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 microm) or intracellular Cl- concentrations (4-145 mm). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.

Amino Acid Transport Systems, Acidic↗

PubMed2004

Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites.

Vesicular glutamate transporters (VGLUTs) 1 and 2 show a mutually exclusive distribution in the adult brain that suggests specialization for synapses with different properties of release. Consistent with this distribution, inactivation of the VGLUT1 gene silenced a subset of excitatory neurons in the adult. However, the same cell populations exhibited VGLUT1-independent transmission early in life. Developing hippocampal neurons transiently coexpressed VGLUT2 and VGLUT1 at distinct synaptic sites with different short-term plasticity. The loss of VGLUT1 also reduced the reserve pool of synaptic vesicles. Thus, VGLUT1 plays an unanticipated role in membrane trafficking at the nerve terminal.

Animals↗

PubMed2004

Electrochemical monitoring of transport by a vesicular monoamine transporter expressed in Xenopus oocytes.

Xenopus laevis oocytes were injected with synthetic mRNA coding for a rat VMAT2 mutant (rVMAT2-I483A/L484A) shown previously to be retained on the plasma membrane as a result of a presumed reduction of endocytosis. Binding of the specific VMAT inhibitor [3H]dihydrotetrabenazine indicated that expression did occur at a level of approximately 3 fmol per oocyte. To determine if rVMAT2-I483A/L484A expressed in oocytes was capable of substrate transport, oocytes were placed in buffer at pH 6.0, dopamine substrate was injected into the cell, and egress of substrate was monitored by fast scan cyclic voltammetry using a carbon fiber microelectrode. Under these conditions, transport by oocytes injected with RNA coding for rVMAT2-I483A/L484A ranged from approximately 0.5 to more than 2.5 pmol/min. Water-injected and uninjected control oocytes did not exhibit appreciable transport activity. Transport by rVMAT2-I483A/L484A-injected oocytes was reduced to control levels by tetrabenazine, a known inhibitor of VMAT transport activity. Comparison of subtracted voltammograms obtained from transport assays with those for calibration experiments confirmed that the transported species was dopamine. These results suggest that expression of VMATs in oocytes may provide a useful model system for mechanistic and regulatory studies that would not be feasible using traditional methods.

Animals↗

PubMed2004

VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate.

Exocytotic release of the excitatory neurotransmitter glutamate depends on transport of this amino acid into synaptic vesicles. Recent work has identified a distinct family of proteins responsible for vesicular glutamate transport (VGLUTs) that show no sequence similarity to the other two families of vesicular neurotransmitter transporters. The distribution of VGLUT1 and VGLUT2 accounts for the ability of most established excitatory neurons to release glutamate by exocytosis. Surprisingly, they show a striking complementary pattern of expression in adult brain that might reflect differences in membrane trafficking. By contrast, VGLUT3 is expressed by many cells traditionally considered to release a different classical transmitter, suggesting novel roles for glutamate as an extracellular signal. VGLUT3 also differs from VGLUT1 and VGLUT2 in its subcellular location, with somatodendritic as well as axonal expression.

Amino Acid Transport System X-AG↗

PubMed2004

The effect of rare human sequence variants on the function of vesicular monoamine transporter 2.

The extent to which genetic variation in a population contributes to phenotypic variation depends on the frequency of sequence polymorphisms and the effect of these polymorphisms on function. The frequency of polymorphisms might also reflect the severity of their effects on function. We therefore examined the effect of very rare single nucleotide polymorphisms (SNPs) on the activity of the vesicular monoamine transporter 2 (VMAT2, SLC18A2), a gene implicated in neuropsychiatric disease. Of the two rare SNPs identified in an ethnically diverse population, neither eliminates transport, but one that involves replacement of a highly conserved residue with a very similar amino acid impairs substrate recognition. This variant, and another affecting an unconserved residue, also affect inhibition by the clinically used drug reserpine. Because VMAT2 influences a form of toxicity similar to Parkinson's disease, we extended the analysis to two SNPs identified in a population with Parkinson's disease. These two SNPs have no detectable effect on most aspects of VMAT2 function, but one that affects a highly conserved residue may increase sensitivity to the inhibitor tetrabenazine. The results illustrate the relationship between conservation of the affected residue, the nature of the substitution and effects on substrate versus inhibitor interaction.

Adrenergic Uptake Inhibitors↗

PubMed2003

Complementary distribution of vesicular glutamate transporters 1 and 2 in the nucleus accumbens of rat: Relationship to calretinin-containing extrinsic innervation and calbindin-immunoreactive neurons.

The caudomedial shell of the rat nucleus accumbens exhibits inhomogeneous distribution patterns of the vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). This paper focuses on the question of whether patterns of VGLUT1 and VGLUT2 correspond to cytoarchitectonically and cytochemically defined subterritories of the caudomedial shell region. VGLUT2 was shown to be coexpressed with calretinin in the dense axonal plexus known to emanate from the paraventricular thalamic nucleus. In regions termed corridors, which are spared by this paraventricular thalamic innervation, axonal terminals were found to be clustered and VGLUT1-immunoreactive. It is assumed that these fibers originate in the prelimbic cortex and/or in the parvicellular basal amygdaloid nucleus known to project to accumbal shell components. Our findings confirm the existence of two well-separated neuronal circuits in the caudomedial shell that are dominated by two different excitatory input systems originating from either thalamic, cortical, or cortex-like amygdaloid sources. The large lateral corridors-which resemble the accumbal core not only in respect to their VGLUT1 immunolabeling but also concerning their content of calbindin-positive cells-may represent a component of the anatomically weakly defined accumbal shore region.

Afferent Pathways↗

PubMed2003

Organic anion transport is the primary function of the SLC17/type I phosphate transporter family.

Recently, molecular studies have determined that the SLC17/type I phosphate transporters, a family of proteins initially characterized as phosphate carriers, mediate the transport of organic anions. While their role in phosphate transport remains uncertain, it is now clear that the transport of organic anions facilitated by this family of proteins is involved in diverse processes ranging from the vesicular storage of the neurotransmitter glutamate to the degradation and metabolism of glycoproteins.

Animals↗

PubMed2003

Postnatal development of the glutamate vesicular transporter VGLUT1 in rat cerebral cortex.

The expression of the vesicular glutamate transporter VGLUT1 in the rat neocortex was studied during postnatal development using immunocytochemistry and Western blotting. At all ages, VGLUT immunoreactivity is localized to puncta that coexpress the presynaptic marker synaptophysin. VGLUT1 immunoreactivity is faint at birth, increases in the subplate during the first postnatal week, invades the supragranular layers in the second week and reaches the adult pattern at P20-P30. Its spatial and temporal maturation patterns suggest that VGLUT1 may be the vesicular transporter in developing corticocortical connections.

Aging↗

PubMed2003

The H+-coupled electrogenic lysosomal amino acid transporter LYAAT1 localizes to the axon and plasma membrane of hippocampal neurons.

Recent work has identified a lysosomal protein that transports neutral amino acids (LYAAT1). We now show that LYAAT1 mediates H+ cotransport with a stoichiometry of 1 H+/1 amino acid, consistent with a role in the active efflux of amino acids from lysosomes. In neurons, however, LYAAT1 localizes to axonal processes as well as lysosomes. In axons LYAAT1 fails to colocalize with synaptic markers. Rather, axonal LYAAT1 colocalizes with the exocyst, suggesting a role for membranes expressing LYAAT1 in specifying sites for exocytosis. A protease protection assay and measurements of intracellular pH further indicate abundant expression at the plasma membrane, raising the possibility of physiological roles for LYAAT1 on the cell surface as well as in lysosomes.

Amino Acid Sequence↗

PubMed2003

Vesicular neurotransmitter transporter expression in developing postnatal rodent retina: GABA and glycine precede glutamate.

Vesicular transporters regulate the amount and type of neurotransmitter sequestered into synaptic vesicles and, hence, the kind of signal transmitted to postsynaptic neurons. Glutamate is the prominent excitatory neurotransmitter in retina; GABA and glycine are the main inhibitory neurotransmitters. Little is known about the ontogeny of vesicular neurotransmission in retina. We investigated expression of glutamatergic [vesicular glutamate transporter 1 (VGLUT1)] and GABA/glycinergic [vesicular GABA/glycine transporter (VGAT)] vesicular transporters in postnatal retina. VGLUT1 labels glutamatergic synapses. VGLUT1 and synaptic vesicle 2 colocalized to photoreceptor terminals. VGLUT1 colocalized with PKC to rod bipolar terminals and to ON bipolar terminals in metabotropic glutamate receptor 6+/- mice. Developmentally, VGAT expression precedes VGLUT1. In rat and mouse retina, VGAT occurred in the inner retina by postnatal day 1 (P1). In rat retina, VGLUT1 was in the outer retina by P5-P7 and the inner retina by P7. In the mouse retina, VGLUT1 expression was in the outer retina by P3 and the inner retina by P5. Both rat and mouse retina had an adult pattern of VGLUT1 expression by P14. VGLUT1 expression precedes ribbon synapses, which are first observed in the inner retina at P11 (Fisher, 1979) in mouse and P13 (Horsburgh and Sefton, 1987) in rat. The ribbon synapse marker RIBEYE was not detected in inner retina of P5 or P7 rat. Spontaneous EPSCs in mouse ganglion cells were recorded as early as P7. Together, these findings indicate that vesicular GABA and glycine transmission precedes vesicular glutamate transmission in developing rodent retina. Furthermore, vesicular glutamate transmission likely occurs before ribbon synapse formation in the inner retina.

Age Factors↗

PubMed2003

Expression of plasma membrane GABA transporters but not of the vesicular GABA transporter in dentate granule cells after kainic acid seizures.

Kainic acid-induced seizures cause a marked increase in the expression of glutamate decarboxylase 67 (GAD67) in granule cells of the dentate gyrus. To determine the possible modes of sequestration of newly formed gamma-aminobutyric acid (GABA), we used in situ hybridization and immunocytochemistry to investigate the expression of several proteins related to GABA in dentate granule cells of rats 4 h to 60 days after kainic acid-induced status epilepticus and in controls. GAD67 and GAD65 mRNA levels were increased by up to 300% and 800%, respectively, in the granule cell layer 6-24 h after kainate injection. Subsequently, increased GAD and GABA immunoreactivity was observed in the terminal field of mossy fibers and in presumed dendrites of granule cells. mRNA of both known plasma membrane GABA transporters (GAT-1 and GAT-3) was expressed in granule cells of control rats. GAT-1 mRNA levels increased (by 30%) 9 h after kainate injection but were reduced by about 25% at later intervals. GAT-3 mRNA was reduced (by 35-75%) in granule cells 4 h to 30 days after kainic acid injection. In contrast, no expression of the mRNA or immunoreactivity of the vesicular GABA transporter was detected in granule cells or in mossy fibers, respectively. GABA transaminase mRNA was only faintly expressed in granule cells, and its levels were reduced (by 60-65%) 12 h to 30 days after kainate treatment. The results indicate that GABA can be taken up and synthesized in granule cells. No evidence for the expression of the vesicular GABA transporter (VGAT) in granule cells was obtained. After sustained epileptic seizures, the markedly increased expression of glutamate decarboxylase and the reduced expression of GABA transaminase may result in increased cytoplasmic GABA concentrations in granule cells. It is suggested that, during epileptic seizures, elevated intracellular GABA and sodium concentration could then result in nonvesicular release of GABA from granule cell dendrites. GABA could then act on GABA-A receptors, protecting granule cells from overexcitation.

Animals↗

PubMed2003

Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root ganglia.

Glutamate is transported into synaptic vesicles by vesicular glutamate transporter (VGLUT) proteins. Three different VGLUTs, VGLUT1, VGLUT2, and VGLUT3, have recently been characterized, and they are considered to represent the most specific marker so far for neurons using glutamate as transmitter. We analyzed the cellular localization of VGLUT1-3 in the rat spinal cord and dorsal root ganglia (DRGs) in control rats and after dorsal rhizotomy. Using in situ hybridization, VGLUT1 mRNA containing neurons were shown in the dorsomedial part of the intermediate zone, whereas VGLUT2 mRNA-expressing neurons were present in the entire intermediate zone, both populations most likely representing interneurons. VGLUT3 mRNA could not be detected in the spinal cord. In the ventral horn, a dense plexus of VGLUT1-immunoreactive (ir) nerve terminals was present, with large varicosities abutting on presumed motoneurons. In the dorsal horn a similarly dense plexus was seen, except in laminae I and II. A very dense plexus of VGLUT2-ir fibers was distributed in the entire gray matter of the spinal cord, with many fibers lying close to presumed motoneurons. Few VGLUT3-ir fibers were distributed in the white and gray matter, including lamina IX. However, a dense VGLUT3-ir plexus was seen in the sympathetic intermedio-lateral column (IML). Multiple-labeling immunohistochemistry revealed that the VGLUT1-, VGLUT2-, and VAChT-containing varicosities in lamina IX all represent separate entities. There was no colocalization of VGLUT3 with VAChT or 5-HT in varicose fibers of the ventral horn, but some VGLUT3-ir fibers in the IML were 5-HT-positive. Lesioning of the dorsal roots resulted in an almost complete disappearance of VGLUT1-ir fibers around motoneurons and a less pronounced decrease in the remaining gray matter, whereas the density of VGLUT2- and VAChT-ir fibers appeared unaltered after lesion. Many VGLUT1-ir neurons were observed in DRGs; they were almost all large and did not colocalize calcitonin gene-related peptide (CGRP), and there was no overlap between these markers in fibers in the superficial dorsal horn. VGLUT2 was, at most, seen in a few DRG neurons. Taken together, these results suggest that the VGLUTs mRNAs are present in distinct subsets of neuronal populations at the spinal level. VGLUT1 is mainly present in primary afferents from large, CGRP-negative DRG neurons, VGLUT2 has mainly a local origin, and VGLUT3 fibers probably have a supraspinal origin.

Amino Acid Transport Systems, Acidic↗

PubMed2003

Plasma membrane and vesicular glutamate transporter mRNAs/proteins in hypothalamic neurons that regulate body weight.

After synaptic release, glutamate is taken up by the nerve terminal via a plasma membrane-associated protein termed excitatory amino acid transporter 3 (EAAT3). Following entry into the nerve terminal, glutamate is pumped into synaptic vesicles by a vesicular transport system. Three different vesicular glutamate transporter proteins (VGLUT1-3) representing unique markers for glutamatergic neurons were recently characterized. The presence of EAAT3, glutaminase and VGLUT1-3 was examined in mouse, rat and rabbit species at mRNA and protein levels in hypothalamic neurons which are involved in the regulation of body weight using in situ hybridization and immunohistochemistry. EAAT3 and glutaminase mRNAs were demonstrated in all parts of the arcuate nucleus in the dorsomedial and ventromedial hypothalamic nuclei and lateral hypothalamic area. VGLUT1 mRNA was present in the magnocellular lateral hypothalamic nucleus. VGLUT2 mRNA was demonstrated in a subpopulation of neurons in the arcuate nucleus and in the ventromedial and dorsomedial hypothalamic nuclei and lateral hypothalamic area. Few VGLUT3 mRNA expressing neurons were scattered throughout the medial and lateral hypothalamus. EAAT3-like immunoreactivity (-li) was demonstrated in glutamate, neuropeptide Y (NPY), agouti-related peptide (AGRP), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), melanin-concentrating hormone and orexin-immunoreactive (-ir) neurons. VGLUT2-li could only be demonstrated in POMC- and CART-ir neurons of the ventrolateral arcuate nucleus. The results show that key neurons involved in regulation of energy balance are glutamatergic and/or densely innervated by glutamatergic nerve terminals. Whereas orexigenic NPY/AGRP neurons situated in the ventromedial part of the arcuate nucleus are mainly GABAergic, it is shown that several anorexigenic POMC/CART neurons of the ventromedial arcuate nucleus are most likely glutamatergic [corrected].

Agouti-Related Protein↗

PubMed2003

Complementary distribution of type 1 cannabinoid receptors and vesicular glutamate transporter 3 in basal forebrain suggests input-specific retrograde signalling by cholinergic neurons.

Basal forebrain cholinergic neurons project to diverse cortical and hippocampal areas and receive reciprocal projections therefrom. Maintenance of a fine-tuned synaptic communication between pre- and postsynaptic cells in neuronal circuitries also requires feedback mechanisms to control the probability of neurotransmitter release from the presynaptic terminal. Release of endocannabinoids or glutamate from a postsynaptic neuron has been identified as a means of retrograde synaptic signalling. Presynaptic action of endocannabinoids is largely mediated by type 1 cannabinoid (CB1) receptors, while fatty-acid amide hydrolase (FAAH) is involved in inactivating some endocannabinoids postsynaptically. Alternatively, vesicular glutamate transporter 3 (VGLUT3) controls release of glutamate from postsynaptic cells. Here, we studied the distribution of CB1 receptors, FAAH and VGLUT3 in cholinergic basal forebrain nuclei of mouse and rat. Cholinergic neurons were devoid of CB1 receptor immunoreactivity. A fine CB1 receptor-immunoreactive (ir) fibre meshwork was present in medial septum, diagonal bands and nucleus basalis. In contrast, the ventral pallidum and substantia innominata received dense CB1 receptor-ir innervation and cholinergic neurons received CB1 receptor-ir presumed synaptic contacts. Consistent with CB1 receptor distribution, FAAH-ir somata were abundant in basal forebrain and appeared in contact with CB1 receptor-containing terminals. Virtually all cholinergic neurons were immunoreactive for FAAH. A significant proportion of cholinergic cells exhibited VGLUT3 immunoreactivity in medial septum, diagonal bands and nucleus basalis, and were in close apposition to VGLUT3-ir terminals. VGLUT3 immunoreactivity was largely absent in ventral pallidum and substantia innominata. We propose that specific subsets of cholinergic neurons may utilize endocannabinoids or glutamate for retrograde control of the efficacy of input synapses, and the mutually exclusive complementary distribution pattern of CB1 receptor-ir and VGLUT3-ir fibres in basal forebrain suggests segregated input-specific signalling mechanisms by cholinergic neurons.

Amidohydrolases↗

PubMed2002

The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate.

Quantal release of the principal excitatory neurotransmitter glutamate requires a mechanism for its transport into secretory vesicles. Within the brain, the complementary expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the release of glutamate by all known excitatory neurons. We now report the identification of VGLUT3 and its expression by many cells generally considered to release a classical transmitter with properties very different from glutamate. Remarkably, subpopulations of inhibitory neurons as well as cholinergic interneurons, monoamine neurons, and glia express VGLUT3. The dendritic expression of VGLUT3 by particular neurons also indicates the potential for retrograde synaptic signaling. The distribution and subcellular location of VGLUT3 thus suggest novel modes of signaling by glutamate.

Amino Acid Transport Systems, Acidic↗

PubMed2002

Methamphetamine-induced degeneration of dopaminergic neurons involves autophagy and upregulation of dopamine synthesis.

Methamphetamine (METH) selectively injures the neurites of dopamine (DA) neurons, generally without inducing cell death. It has been proposed that METH-induced redistribution of DA from the vesicular storage pool to the cytoplasm, where DA can oxidize to produce quinones and additional reactive oxygen species, may account for this selective neurotoxicity. To test this hypothesis, we used mice heterozygous (+/-) or homozygous (-/-) for the brain vesicular monoamine uptake transporter VMAT2, which mediates the accumulation of cytosolic DA into synaptic vesicles. In postnatal ventral midbrain neuronal cultures derived from these mice, METH-induced degeneration of DA neurites and accumulation of oxyradicals, including metabolites of oxidized DA, varied inversely with VMAT2 expression. METH administration also promoted the synthesis of DA via upregulation of tyrosine hydroxylase activity, resulting in an elevation of cytosolic DA even in the absence of vesicular sequestration. Electron microscopy and fluorescent labeling confirmed that METH promoted the formation of autophagic granules, particularly in neuronal varicosities and, ultimately, within cell bodies of dopaminergic neurons. Therefore, we propose that METH neurotoxicity results from the induction of a specific cellular pathway that is activated when DA cannot be effectively sequestered in synaptic vesicles, thereby producing oxyradical stress, autophagy, and neurite degeneration.

Animals↗