A subset of ventral tegmental area dopamine neurons responds to acute ethanol.

The mechanisms by which alcohol drinking promotes addiction in humans and self-administration in rodents remain obscure, but it is well known that alcohol can enhance dopamine (DA) neurotransmission from neurons of the ventral tegmental area (VTA) and increase DA levels within the nucleus accumbens and prefrontal cortex. We recorded from identified DA neuronal cell bodies within ventral midbrain slices prepared from a transgenic mouse line (TH-GFP) using long-term stable extracellular recordings in a variety of locations and carefully mapped the responses to applied ethanol (EtOH). We identified a subset of DA neurons in the medial VTA located within the rostral linear and interfascicular nuclei that fired spontaneously and exhibited a concentration-dependent increase of firing frequency in response to EtOH, with some neurons responsive to as little as 20mM EtOH. Many of these medial VTA DA neurons were also insensitive to the D2 receptor agonist quinpirole. In contrast, DA neurons in the lateral VTA (located within the parabrachial pigmented and paranigral nuclei) were either unresponsive or responded only to 100mM EtOH. Typically, these lateral VTA DA cells had very slow firing rates, and all exhibited inhibition by quinpirole via D2 "autoreceptors". VTA non-DA cells did not show any significant response to low levels of EtOH. These findings are consistent with evidence for heterogeneity among midbrain DA neurons and provide an anatomical and pharmacological distinction between DA neuron sub-populations that will facilitate future mechanistic studies on the actions of EtOH in the VTA.

Dopamine-dependent corticostriatal synaptic filtering regulates sensorimotor behavior.

Modulation of corticostriatal synaptic activity by dopamine is required for normal sensorimotor behaviors. After loss of nigrostriatal dopamine axons in Parkinson's disease, l-3,4-dihydroxyphenlalanine (l-DOPA) and dopamine D2-like receptor agonists are used as replacement therapy, although these drugs also trigger sensitized sensorimotor responses including dyskinesias and impulse control disorders. In mice, we lesioned dopamine projections to the left dorsal striatum and assayed unilateral sensorimotor deficits with the corridor test as well as presynaptic corticostriatal activity with the synaptic vesicle probe, FM1-43. Sham-lesioned mice acquired food equivalently on both sides, while D2 receptor activation filtered the less active corticostriatal terminals, a response that required coincident co-activation of mGlu-R5 metabotropic glutamate and CB1 endocannabinoid receptors. Lesioned mice did not acquire food from their right, but overused that side following treatment with l-DOPA. Synaptic filtering on the lesioned side was abolished by either l-DOPA or a D2 receptor agonist, but when combined with a CB1 receptor antagonist, l-DOPA or D2 agonists normalized both synaptic filtering and behavior. Thus, high-pass filtering of corticostriatal synapses by the coordinated activation of D2, mGlu-R5, and CB1 receptors is required for normal sensorimotor response to environmental cues.

The neuromelanin of human substantia nigra: structure, synthesis and molecular behaviour.

The pigmented neurons of the substantia nigra (SN) are typically lost in Parkinson's disease: however the possible relationship between neuronal vulnerability and the presence of neuromelanin (NM) has not been elucidated. Early histological studies revealed the presence of increasing amounts of NM in the SN with aging in higher mammals, showed that NM granules are surrounded by membrane, and comparatively evaluated the pigmentation of SN in different animal species. Histochemical studies showed the association of NM with lipofuscins. However, systematic investigations of NM structure, synthesis and molecular interactions have been undertaken only during the last decade. In these latter studies, NM was identified as a genuine melanin with a strong chelating ability for iron and affinity for compounds such as lipids, pesticides, and MPP+. The affinity of NM for a variety of inorganic and organic toxins is consistent with a postulated protective function for NM. Moreover, the neuronal accumulation of NM during aging, and the link between its synthesis and high cytosolic concentration of catechols suggests a protective role. However, its putative neuroprotective effects could be quenched in conditions of toxin overload.

Autophagy in neurons: a review.

Macroautophagy is a process of regulated turnover of cellular constituents that occurs during development and under conditions of stress such as starvation. Defects in autophagy have serious consequences, as they have been linked to neurodegenerative disease, cancer, and cardiomyopathy. This process, which exists in all eukaryotic cells, is tightly controlled, but in extreme cases results in the death of the cell. While major insights into the molecular and biochemical pathways involved have come from genetic studies in yeast, little is known about autophagic pathways in mammalian cells, particularly in neurons. Recently, research in neuronal culture models has begun to identify some characteristics of neuronal macroautophagy. The results suggest that macroautophagy in neurons may provide a neuroprotective mechanism. Here, we review the defining characteristics of autophagy with special attention to its role in neurodegenerative disorders, and recent efforts to delineate the pathway of autophagic protein degradation in neurons.

The neuromelanin of human substantia nigra and its interaction with metals.

Neuromelanin (NM) is a peculiar biochemical component of several neurons in the Substantia Nigra (SN), the target area of the degenerative process in Parkinson Disease (PD). SN NM has peculiarities as to its composition and an impressive capacity of chelating metals, iron in particular, but not exclusively. Gaining insights into the structural and functional characteristics of NM should help understanding the reasons of selective vulnerability of nigral neurons in many parkinsonian conditions. From the present data a protective role of NM can be postulated until the buffering capability toward heavy metals are exhausted. The overloading of NM with iron and other metals in neurons may trigger inflammatory and degenerative processes aggravating the underlying pathological condition.

Expression of A53T mutant but not wild-type alpha-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death.

Alpha-synuclein mutations have been identified in certain families with Parkinson's disease (PD), and alpha-synuclein is a major component of Lewy bodies. Other genetic data indicate that the ubiquitin-dependent proteolytic system is involved in PD pathogenesis. We have generated stable PC12 cell lines expressing wild-type or A53T mutant human alpha-synuclein. Lines expressing mutant but not wild-type alpha-synuclein show: (1) disruption of the ubiquitin-dependent proteolytic system, manifested by small cytoplasmic ubiquitinated aggregates and by an increase in polyubiquitinated proteins; (2) enhanced baseline nonapoptotic death; (3) marked accumulation of autophagic-vesicular structures; (4) impairment of lysosomal hydrolysis and proteasomal function; and (5) loss of catecholamine-secreting dense core granules and an absence of depolarization-induced dopamine release. Such findings raise the possibility that the primary abnormality in these cells may involve one or more deficits in the lysosomal and/or proteasomal degradation pathways, which in turn lead to loss of dopaminergic capacity and, ultimately, to death. These cells may serve as a model to study the effects of aberrant alpha-synuclein on dopaminergic cell function and survival.

Substantia nigra neuromelanin: structure, synthesis, and molecular behaviour.

The pigmented neurones of the substantia nigra are typically lost in Parkinson's disease; however, the possible relation between neuronal vulnerability and the presence of neuromelanin has not been elucidated. Early histological studies revealed the presence of increasing amounts of neuromelanin in the substantia nigra with aging in higher mammals, showed that the neuromelanin granules are surrounded by a membrane, and comparatively evaluated the pigmentation of the substantia nigra in different animal species. Histochemical studies showed the association of neuromelanin with lipofuscins. However, systematic investigations of the structure, synthesis, and molecular interactions of neuromelanin have been undertaken only during the past decade. In these later studies, neuromelanin was identified as a genuine melanin with a strong chelating ability for iron and an affinity for compounds such as lipids, pesticides, and MPP(+). The affinity of neuromelanin for a variety of inorganic and organic toxins is consistent with a postulated protective function for neuromelanin. Moreover, the neuronal accumulation of neuromelanin during aging and the link between its synthesis and a high cytosolic concentration of catechols suggest a protective role. However, its putative neuroprotective effects could be quenched in conditions of toxin overload.

Brain-derived neurotrophic factor inhibits apoptosis and dopamine-induced free radical production in striatal neurons but does not prevent cell death.

In hereditary Huntington's disease, a triplet repeat disease, there is extensive loss of striatal neurons. It has been shown that brain-derived neurotrophic factor (BDNF) protects striatal neurons against a variety of insults. We confirmed that BDNF enhances survival and DARPP-32 expression in primary striatal cultures derived from postnatal mice. Furthermore, BDNF inhibited intracellular oxyradical stress triggered by dopamine, and partially blocked basal and dopamine-induced apoptosis. Nevertheless, BDNF failed to rescue striatal neurons from dopamine-induced cell death. Therefore, BDNF inhibits free radical and apoptotic pathways in medium spiny neurons, but does so downstream from the point of commitment to cell death.

Mice transgenic for exon 1 of the Huntington's disease gene display reduced striatal sensitivity to neurotoxicity induced by dopamine and 6-hydroxydopamine.

Huntington's disease is an autosomal dominant hereditary neurodegenerative disorder characterized by severe striatal cell loss. Dopamine (DA) has been suggested to play a role in the pathogenesis of the disease. We have previously reported that transgenic mice expressing exon 1 of the human Huntington gene (R6 lines) are resistant to quinolinic acid-induced striatal toxicity. In this study we show that with increasing age, R6/1 and R6/2 mice develop partial resistance to DA- and 6-hydroxydopamine-mediated toxicity in the striatum. Using electron microscopy, we found that the resistance is localized to the cell bodies and not to the neuropil. The reduction of dopamine and cAMP regulated phosphoprotein of a molecular weight of 32 kDa (DARPP-32) in R6/2 mice does not provide the resistance, as DA-induced striatal lesions are not reduced in size in DARPP-32 knockout mice. Neither DA receptor antagonists nor a N-methyl-d-aspartate (NMDA) receptor blocker reduce the size of DA-induced striatal lesions, suggesting that DA toxicity is not dependent upon DA- or NMDA receptor-mediated pathways. Moreover, superoxide dismutase-1 overexpression, monoamine oxidase inhibition and the treatment with the free radical scavenging spin-trap agent phenyl-butyl-tert-nitrone (PBN) also did not block DA toxicity. Levels of the antioxidant molecules, glutathione and ascorbate were not increased in R6/1 mice. Because damage to striatal neurons following intrastriatal injection of 6-hydroxydopamine was also reduced in R6 mice, a yet-to-be identified antioxidant mechanism may provide neuroprotection in these animals. We conclude that striatal neurons of R6 mice develop resistance to DA-induced toxicity with age.

Amphetamine distorts stimulation-dependent dopamine overflow: effects on D2 autoreceptors, transporters, and synaptic vesicle stores.

Amphetamine (AMPH) is known to raise extracellular dopamine (DA) levels by inducing stimulation-independent DA efflux via reverse transport through the DA transporter and by inhibiting DA re-uptake. In contrast, recent studies indicate that AMPH decreases stimulation-dependent vesicular DA release. One candidate mechanism for this effect is the AMPH-mediated redistribution of DA from vesicles to the cytosol. In addition, the inhibition of stimulation-dependent release may occur because of D2 autoreceptor activation by DA that is released via reverse transport. We used the D2 receptor antagonist sulpiride and mice lacking the D2 receptor to address this issue. To evaluate carefully AMPH effects on release and uptake, we recorded stimulated DA overflow in striatal slices by using continuous amperometry and cyclic voltammetry. Recordings were fit by a random walk simulation of DA diffusion, including uptake with Michaelis-Menten kinetics, that provided estimates of DA concentration and uptake parameters. AMPH (10 microm) promoted the overflow of synaptically released DA by decreasing the apparent affinity for DA uptake (K(m) increase from 0.8 to 32 microm). The amount of DA released per pulse, however, was decreased by 82%. This release inhibition was prevented partly by superfusion with sulpiride (47% inhibition) and was reduced in D2 mutant mice (23% inhibition). When D2 autoreceptor activation was minimal, the combined effects of AMPH on DA release and uptake resulted in an enhanced overflow of exocytically released DA. Such enhancement of stimulation-dependent DA overflow may occur under conditions of low D2 receptor activity or expression, for example as a result of AMPH sensitization.

Proteasomal inhibition leads to formation of ubiquitin/alpha-synuclein-immunoreactive inclusions in PC12 cells.

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-tBu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human alpha-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another.

Expanded CAG repeats in exon 1 of the Huntington's disease gene stimulate dopamine-mediated striatal neuron autophagy and degeneration.

Huntington's disease (HD) is caused by an expanded CAG repeat in exon 1 of the gene coding for the huntingtin protein. The cellular pathway by which this mutation induces HD remains unknown, although alterations in protein degradation are involved. To study intrinsic cellular mechanisms linked to the mutation, we examined dissociated postnatally derived cultures of striatal neurons from transgenic mice expressing exon 1 of the human HD gene carrying a CAG repeat expansion. While there was no difference in cell death between wild-type and mutant littermate-derived cultures, the mutant striatal neurons exhibited elevated cell death following a single exposure to a neurotoxic concentration of dopamine. The mutant neurons exposed to dopamine also exhibited lysosome-associated responses including induction of autophagic granules and electron-dense lysosomes. The autophagic/lysosomal compartments co-localized with high levels of oxygen radicals in living neurons, and ubiquitin. The results suggest that the combination of mutant huntingtin and a source of oxyradical stress (provided in this case by dopamine) induces autophagy and may underlie the selective cell death characteristic of HD.

Dale's principle and glutamate corelease from ventral midbrain dopamine neurons.

While direct application of dopamine modulates postsynaptic activity, electrical stimulation of dopamine neurons typically evokes excitation. Most of this excitation appears to be due to activation of collateral pathways; however, several lines of evidence have suggested that there is a monosynaptic component due to glutamate corelease by dopamine neurons. Recently, more direct evidence obtained in culture has shown that ventral midbrain dopamine neurons release both dopamine and glutamate. Moreover, they appear to do so from separate release sites, calling into question recent modifications of Dale's Principle. The neurochemical phenotype of a given synapse may be determined by subcellular neurotransmitter levels, uptake, or storage. However, the relationship between dopamine and glutamate release from dopamine neuron synapses in the intact brain--and the mechanisms involved--has yet to be resolved.

Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles.

Melanin, the pigment in hair, skin, eyes, and feathers, protects external tissue from damage by UV light. In contrast, neuromelanin (NM) is found in deep brain regions, specifically in loci that degenerate in Parkinson's disease. Although this distribution suggests a role for NM in Parkinson's disease neurodegeneration, the biosynthesis and function of NM have eluded characterization because of lack of an experimental system. We induced NM in rat substantia nigra and PC12 cell cultures by exposure to l-dihydroxyphenylalanine, which is rapidly converted to dopamine (DA) in the cytosol. This pigment was identical to human NM as assessed by paramagnetic resonance and was localized in double membrane autophagic vacuoles identical to NM granules of human substantia nigra. NM synthesis was abolished by adenoviral-mediated overexpression of the synaptic vesicle catecholamine transporter VMAT2, which decreases cytosolic DA by increasing vesicular accumulation of neurotransmitter. The NM is in a stable complex with ferric iron, and NM synthesis was inhibited by the iron chelator desferrioxamine, indicating that cytosolic DA and dihydroxyphenylalanine are oxidized by iron-mediated catalysis to membrane-impermeant quinones and semiquinones. NM synthesis thus results from excess cytosolic catecholamines not accumulated into synaptic vesicles. The permanent accumulation of excess catechols, quinones, and catechol adducts into a membrane-impermeant substance trapped in organelles may provide an antioxidant mechanism for catecholamine neurons. However, NM in organelles associated with secretory pathways may interfere with signaling, as it delays stimulated neurite outgrowth in PC12 cells.

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Regulation of quantal size by presynaptic mechanisms.

Quantal size is often modeled as invariant, although it is now well established that the number of transmitter molecules released per synaptic vesicle during exocytosis can be modulated in central and peripheral synapses. In this review, we suggest why presynaptically altered quantal size would be important at social synapses that provide extrasynaptic neurotransmitter. Current techniques used to measure quantal size are reviewed with particular attention to amperometry, the first approach to provide direct measurement of the number of molecules and kinetics of presynaptic quantal release, and to CNS dopamine neuronal terminals. The known interventions that alter quantal size at the presynaptic locus are reviewed and categorized as (1) alteration of transvesicular free energy gradients, (2) modulation of vesicle transmitter transporter activity, (3) modulation of fusion pore kinetics, (4) altered transmitter degranulation, and (5) changes in synaptic vesicle volume. Modulation of the number of molecules released per quantum underlies mechanisms of drug action of L-DOPA and the amphetamines, and seems likely to be involved in both normal synaptic modification and disease states. Statistical analysis for examining quantal size and data presentation is discussed. We include detailed information on performing nonparametric resampling statistical analysis, the Kolmogorov-Smirnov test for two populations, and random walk simulations using spreadsheet programs.

Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system.

alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinson's disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.