Search

In Parkinson’s disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA−/− neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker.

Dopaminergic neurons modulate neural circuits and behaviors via dopamine (DA) release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell-type specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find that most proteins encoded by DA neuron-enriched genes are localized within striatal dopaminergic axons, including ion channels with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.

Optical imaging of changes in membrane potential of living cells can be achieved by the means of fluorescent voltage sensitive dyes (VSDs). A particularly challenging task is to efficiently deliver these highly lipophilic probes to specific neuronal subpopulations in brain tissue. We have tackled this task by designing a solubilizing, hydrophilic polymer platform that carries a high-affinity ligand for a membrane protein marker of interest and a fluorescent VSD. Here, we disclose an improved design of polymer supported probes for chemical, non-genetic targeting of voltage sensors to axons natively expressing the dopamine transporter in ex vivo mouse brain tissue. We first show that for negatively charged rhodol VSDs functioning on the photoinduced electron transfer principle, poly(ethylene glycol) (PEG) as a carrier enables targeting with higher selectivity than the polysaccharide dextran in HEK cell culture. In the same experimental setting, we also demonstrate that incorporation of an azetidine ring in the rhodol chromophore substantially increases the brightness and voltage sensitivity of the respective VSD. We show that the superior properties of the optimized sensor are transferable to recording of electrically evoked activity from dopaminergic axons in mouse striatal slices after averaging of multiple trials. Finally, we suggest the next milestones for the field to achieve single-scan recordings with non-genetically targeted VSDs in native brain tissue.

SummaryThe impact of long-term opioid exposure on the embryonic brain is crucial to healthcare due to the surging number of pregnant mothers with an opioid dependency. Current studies on the neuronal effects are limited due to human brain inaccessibility and cross-species differences among animal models. Here, we report a model to assess cell-type specific responses to acute and chronic fentanyl treatment, as well as fentanyl withdrawal, using human induced pluripotent stem cell (hiPSC)-derived midbrain organoids. Single cell mRNA sequencing (25,510 single cells in total) results suggest that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters the pathways associated with synaptic activities and neuron projection. Acute fentanyl treatment, however, increases dopamine release but does not induce significant changes in gene expressions of cell lineage development. To date, our study is the first unbiased examination of midbrain transcriptomics with synthetic opioid treatment at the single cell level.

Dopaminergic neurons modulate neural circuits and behaviors via dopamine release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell type-specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find a significant enrichment of proteins encoded by Parkinson’s disease-linked genes in striatal dopaminergic axons, including proteins with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.

Significance We report a long-sought& in vivo& physiological role for& α-synuclein (α-syn) in dopamine signaling. The results indicate that& α-syn is critical for activity-dependent dopamine plasticity, and that short repeated burst activity produces rapid presynaptic facilitation, while prolonged burst activity slowly depresses evoked dopamine release. We propose that the rapid facilitation is due to an enhanced fusion of synaptic vesicles at active zones during exocytosis while the depression is due to synaptic exhaustion. These results identify a& dynamic role of& α-syn, and are critical for defining& molecular mechanisms and therapeutic targets for various neurological disorders, where the firing properties of neurons are severely altered., α-Synuclein is expressed at high levels at presynaptic terminals, but defining its role in the regulation of neurotransmission under physiologically relevant conditions has proven elusive. We report that, in vivo, α-synuclein is responsible for the facilitation of dopamine release triggered by action potential bursts separated by short intervals (seconds) and a depression of release with longer intervals between bursts (minutes). These forms of presynaptic plasticity appear to be independent of the presence of β- and γ-synucleins or effects on presynaptic calcium and are consistent with a role for synucleins in the enhancement of synaptic vesicle fusion and turnover. These results indicate that the presynaptic effects of α-synuclein depend on specific patterns of neuronal activity.

Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson's disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson's disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies.

SummaryChanges in striatal cholinergic interneuron (ChI) activity are thought to contribute to Parkinson’s disease pathophysiology and dyskinesia from chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, but the physiological basis of these changes are unknown. We find that dopamine lesion decreases the spontaneous firing rate of ChIs, whereas chronic treatment with L-DOPA of lesioned mice increases baseline ChI firing rates to levels beyond normal activity. The effect of dopamine loss on ChIs was due to decreased currents of both hyperpolarization-activated cyclic nucleotide-gated (HCN) and small conductance calcium-activated potassium (SK) channels. L-DOPA reinstatement of dopamine normalized HCN activity, but SK current remained depressed. Pharmacological blockade of HCN and SK activities mimicked changes in firing, confirming that these channels are responsible for the molecular adaptation of ChIs to dopamine loss and chronic L-DOPA treatment. These findings suggest that targeting ChIs with channel-specific modulators may provide therapeutic approaches for alleviating L-DOPA-induced dyskinesia in PD patients.

RATIONALE: GLT-1 is the major glutamate transporter in the brain and is expressed predominantly in astrocytes but is also present in excitatory axon terminals. To understand the functional significance of GLT-1 expressed in neurons, we generated a conditional GLT-1 knockout mouse and inactivated GLT-1 in neurons using Cre-recombinase expressed under the synapsin 1 promoter, (synGLT-1 KO). OBJECTIVES: Abnormalities of glutamate homeostasis have been shown to affect hippocampal-related behaviors including learning and memory as well as responses to drugs of abuse. Here, we asked whether deletion of GLT-1 specifically from neurons would affect behaviors that assessed locomotor activity, cognitive function, sensorimotor gating, social interaction, as well as amphetamine-stimulated locomotor activity. METHODS/RESULTS: We found that the neuronal GLT-1 KO mice performed similarly to littermate controls in the behavioral tests we studied. Although performance in open field testing was normal, the acute locomotor response to amphetamine was significantly blunted in the synGLT-1 KO (40% of control). We found no change in amphetamine-stimulated extracellular dopamine in the medial shell of the nucleus accumbens, no change in electrically stimulated or amphetamine-induced dopamine release, and no change in dopamine tissue content. CONCLUSIONS: These results support the view that GLT-1 expression in neurons is required for amphetamine-induced behavioral activation, and suggest that this phenotype is not produced through a change in dopamine uptake or release. Although GLT-1 is highly expressed in neurons in the CA3 region of the hippocampus, the tests used in this study were not able to detect a behavioral phenotype referable to hippocampal dysfunction.

α-Synuclein (α-Syn) is a small cytosolic protein associated with a range of cellular compartments, including synaptic vesicles, the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes. In addition to its physiological role in regulating presynaptic function, the protein plays a central role in both sporadic and familial Parkinson’s disease (PD) via a gain-of-function mechanism. Because of this, several recent strategies propose to decrease α-Syn levels in PD patients. While these therapies may offer breakthroughs in PD management, the normal functions of α-Syn and potential side effects of its depletion require careful evaluation. Here, we review recent evidence on physiological and pathological roles of α-Syn in regulating activity-dependent signal transduction and gene expression pathways that play fundamental role in synaptic plasticity.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer’s disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

Altered prefrontal cortex function is implicated in schizophrenia (SCZ) pathophysiology and could arise from imbalance between excitation and inhibition (E/I) in local circuits. It remains unclear whether and how such imbalances relate to genetic etiologies. We used a mouse model of the SCZ-predisposing 22q11.2 deletion (Df(16)A(+/−) mice) to evaluate how this genetic lesion affects the excitability of layer V prefrontal pyramidal neurons and its modulation by dopamine (DA). Df(16)A(+/)(−) mice have normal balance between E/I at baseline but are unable to maintain it upon dopaminergic challenge. Specifically, in wild-type mice, D1 receptor (D1R) activation enhances excitability of layer V prefrontal pyramidal neurons and D2 receptor (D2R) activation reduces it. Whereas the excitatory effect upon D1R activation is enhanced in Df(16)A(+/−) mice, the inhibitory effect upon D2R activation is reduced. The latter is partly due to the inability of mutant mice to activate GABAergic parvalbumin (PV)+ interneurons through D2Rs. We further demonstrate that reduced KCNQ2 channel function in PV+ interneurons in Df(16)A(+/−) mice renders them less capable of inhibiting pyramidal neurons upon D2 modulation. Thus, DA modulation of PV+ interneurons and control of E/I are altered in Df(16)A(+/−) mice with a higher excitation and lower inhibition during dopaminergic modulation.

Dopamine neurotransmission in the dorsal hippocampus is critical for a range of functions from spatial learning and synaptic plasticity to the deficits underlying psychiatric disorders such as attention-deficit hyperactivity disorder. The ventral tegmental area (VTA) is the presumed source of dopamine in the dorsal hippocampus. However, there is a surprising scarcity of VTA dopamine axons in the dorsal hippocampus despite the dense network of dopamine receptors. We have explored this apparent paradox using optogenetic, biochemical, and behavioral approaches and found that dopaminergic axons and subsequent dopamine release in the dorsal hippocampus originate from neurons of the locus coeruleus (LC). Photostimulation of LC axons produced an increase in dopamine release in the dorsal hippocampus as revealed by high-performance liquid chromatography. Furthermore, optogenetically induced release of dopamine from the LC into the dorsal hippocampus enhanced selective attention and spatial object recognition via the dopamine D1/D5 receptor. These results suggest that spatial learning and memory are energized by the release of dopamine in the dorsal hippocampus from noradrenergic neurons of the LC. The present findings are critical for identifying the neural circuits that enable proper attention selection and successful learning and memory.

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.

1-Methyl-4-phenylpyridinium (MPP(+)), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP(+) exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DAcyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP(+) exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP(+) concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP(+) depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DAcyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP(+)-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DAcyt levels in MPP(+)-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in ∼30% reduction of MPP(+)-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP(+) on neuronal DA homeostasis and neurotoxicity.

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, invitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes invivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

Fluoxetine is a widely used antidepressant with an action that is primarily attributed to the inhibition of serotonin re-uptake into the synaptic terminals of the central nervous system. Fluoxetine also has blocking effects on various ion channels, including Ca(2+) channels. It remains unclear, however, how fluoxetine may affect synaptically induced [Ca(2+)](i) spikes. We investigated the effects of fluoxetine on [Ca(2+)](i) spikes, along with the subsequent neurotoxicity that is synaptically evoked by lowering extracellular Mg(2+) in cultured rat hippocampal neurons. Fluoxetine inhibited the synaptically induced [Ca(2+)](i) spikes in p-chloroamphetamine-treated and non-treated neurons, in a concentration-dependent manner. However, other selective serotonin reuptake inhibitors, such as paroxetine and citalopram, did not significantly affect the spikes. Pretreatment with fluoxetine for 5 min inhibited [Ca(2+)](i) increases induced by glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and N-methyl-d-aspartate. Fluoxetine also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. In addition, fluoxetine decreased the [Ca(2+)](i) responses induced by the metabotrophic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine or the ryanodine receptor agonist caffeine. Fluoxetine inhibited [Ca(2+)](i) responses induced by 20mM KCl. Fluoxetine decreased the release of FM1-43 induced by electric field stimulation. Furthermore, fluoxetine inhibited 0.1mM [Mg(2+)](o)-induced cell death. Collectively, our results suggest that fluoxetine suppresses the spikes and protects neurons against excitotoxicity, particularly in cultured rat hippocampal neurons, presumably due to both direct inhibition of presynaptic glutamate release and postsynaptic glutamate receptor-mediated [Ca(2+)](i) signaling. In addition to an indirect inhibitory effect via 5-HT levels, these data suggest a new, possibly direct inhibitory action of fluoxetine on synaptically induced [Ca(2+)](i) spikes and neuronal cell death.

Ethanol is a wildly abused substance that causes various problems and damage in our society. We examined the connection between the action of ethanol and the endocannabinoid system in corticostriatal synaptic transmission by recording excitatory post-synaptic currents (EPSCs). Acute treatment of ethanol (100 mM) inhibited corticostriatal EPSCs. In the presence of AM 251 (5 μM), a cannabinoid 1 (CB1)-receptor antagonist, or AM 404 (5 μM), a cannabinoid transporter inhibitor, the inhibition of corticostriatal EPSCs caused by ethanol was significantly reduced. This result suggests the possibility that the endocannabinoid system is involved in the action of ethanol. To support this result, brain slices were pre-treated with WIN 55,212-2 (1 μM), a CB1-receptor agonist, following treatment of ethanol or treated with WIN 55,212-2 alone. There was no significant difference between each other, indicating that when CB1 receptors are previously activated, the effect of ethanol is blunted. These results suggest that the activation of the endocannabinoid system is one of the possible mechanisms involved in ethanol-induced corticostriatal synaptic depression. Keywords:: striatum, endocannabinoid, ethanol, synaptic depression

P2X4 receptors are calcium-permeable cation channels gated by extracellular ATP. They are found close to subsynaptic sites on hippocampal CA1 neurons. We compared features of synaptic strengthening between wild-type and P2X4 knockout mice (21–26 days old). Potentiation evoked by a tetanic presynaptic stimulus (100 Hz, 1 s) paired with postsynaptic depolarization was less in P2X4−/− mice than in wild-type mice (230 vs. 50% potentiation). Paired-pulse ratios and the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) were not different between wild-type and knockout mice. Prior hyperpolarization (ten 3 s pulses to −120 mV at 0.17 Hz) potentiated the amplitude of spontaneous EPSCs in wild-type mice, but not in P2X4−/− mice; this potentiation was not affected by nifedipine, but was abolished by 10 mm 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA) in the recording pipette. The amplitude of N-methyl-d-aspartate EPSCs (in 6-cyano-7-nitroquinoxaline-2,3-dione, 10 or 30 μm, at −100 mV) facilitated during 20 min recording in magnesium-free solution. In wild-type mice, this facilitation of the N-methyl-d-aspartate EPSC was reduced by about 50% by intracellular BAPTA (10 mm), ifenprodil (3 μm) or 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 μm). In P2X4−/− mice, the facilitation was much less, and was unaffected by intracellular BAPTA, ifenprodil (3 μm) or mitogen-activated protein (MAP) kinase inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 μm). This suggests that the absence of P2X4 receptors limits the incorporation of NR2B subunits into synaptic N-methyl-d-aspartate receptors.

Neural circuits are formed and refined during childhood, including via critical changes in neuronal excitability. Here, we investigated the ontogeny of striatal intrinsic excitability. We found that dopamine neuro-transmission increases from the first to the third postnatal week in mice and precedes the reduction in spiny projection neuron (SPN) intrinsic excitability during the fourth postnatal week. In mice developmentally deficient for striatal dopamine, direct pathway D1-SPNs failed to undergo maturation of excitability past P18 and maintained hyperexcitability into adulthood. We found that the absence of D1-SPN maturation was due to altered phosphatidylinositol 4,5-biphosphate dynamics and a consequent lack of normal ontogenetic increases in Kir2 currents. Dopamine replacement corrected these deficits in SPN excitability when provided from birth or during a specific period of juvenile development (P18–P28), but not during adulthood. These results identify a sensitive period of dopamine-dependent striatal maturation, with implications for the pathophysiology and treatment of neurodevelopmental disorders.

Parkinson’s disease (PD) is a debilitating neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra (SN). Although mitochondrial dysfunction and dysregulated α-synuclein (aSyn) expression are postulated to play a role in PD pathogenesis, it is still debated why neurons of the SN are targeted while neighboring dopaminergic neurons of the ventral tegmental area (VTA) are spared. Using electrochemical and imaging approaches, we investigated metabolic changes in cultured primary mouse midbrain dopaminergic neurons exposed to a parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). We demonstrate that the higher level of neurotoxicity in SN than VTA neurons was due to SN neuron-specific toxin-induced increase in cytosolic dopamine (DA) and Ca2+, followed by an elevation of mitochondrial Ca2+, activation of nitric oxide synthase (NOS), and mitochondrial oxidation. The increase in cytosolic Ca2+was not caused by MPP+-induced oxidative stress, but rather depended on the activity of both L-type calcium channels and aSyn expression, suggesting that these two established pathogenic factors in PD act in concert.

The purpose of this study was to examine the effects of ethanol on synaptic transmission in the dorsal striatum in rat brain slices. The effects of ethanol on corticostriatal synaptic transmission were tested by whole-cell voltage-clamp recording. Ethanol significantly decreased corticostriatal excitatory postsynaptic currents (EPSCs) in a dose-dependent manner (10-200 mM). However, the paired-pulse ratio was not affected by the ethanol (100 mM) treatment. The amplitude of miniature EPSCs (mEPSCs) from these neurons, recorded without cortical stimulation, was decreased, but the frequency of the mEPSCs remained unchanged. Ethanol also decreased currents induced by the local pressure injection of glutamate into dorsal striatal neurons. These results suggest that ethanol inhibits glutamatergic synaptic transmission in the dorsal striatum, possibly through a postsynaptic mechanism.

Recent studies have shown evidence of behavioral recovery after transplantation of human pluripotent stem cell (PSC)-derived neural cells in animal models of neurological disease1–4. However, little is known about the mechanisms underlying graft function. Here we use optogenetics to modulate in real time electrophysiological and neurochemical properties of mesencephalic dopaminergic (mesDA) neurons derived from human embryonic stem cells (hESCs). In mice that had recovered from lesion-induced Parkinsonian motor deficits, light-induced selective silencing of graft activity rapidly and reversibly re-introduced the motor deficits. The re-introduction of motor deficits was prevented by the dopamine agonist apomorphine. These results suggest that functionality depends on graft neuronal activity and dopamine release. Combining optogenetics, slice electrophysiology and pharmacological approaches, we further show that mesDA-rich grafts modulate host glutamatergic synaptic transmission onto striatal medium spiny neurons in a manner reminiscent of endogenous mesDA neurons. Thus, application of optogenetics in cell therapy can link transplantation, animal behavior and postmortem analysis to enable the identification of mechanisms that drive recovery.

Fluoxetine is a widely used antidepressant with an action that is primarily attributed to the inhibition of serotonin re-uptake into the synaptic terminals of the central nervous system. Fluoxetine also has blocking effects on various ion channels, including Ca(2+) channels. It remains unclear, however, how fluoxetine may affect synaptically induced [Ca(2+)](i) spikes. We investigated the effects of fluoxetine on [Ca(2+)](i) spikes, along with the subsequent neurotoxicity that is synaptically evoked by lowering extracellular Mg(2+) in cultured rat hippocampal neurons. Fluoxetine inhibited the synaptically induced [Ca(2+)](i) spikes in p-chloroamphetamine-treated and non-treated neurons, in a concentration-dependent manner. However, other selective serotonin reuptake inhibitors, such as paroxetine and citalopram, did not significantly affect the spikes. Pretreatment with fluoxetine for 5 min inhibited [Ca(2+)](i) increases induced by glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and N-methyl-d-aspartate. Fluoxetine also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. In addition, fluoxetine decreased the [Ca(2+)](i) responses induced by the metabotrophic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine or the ryanodine receptor agonist caffeine. Fluoxetine inhibited [Ca(2+)](i) responses induced by 20mM KCl. Fluoxetine decreased the release of FM1-43 induced by electric field stimulation. Furthermore, fluoxetine inhibited 0.1mM [Mg(2+)](o)-induced cell death. Collectively, our results suggest that fluoxetine suppresses the spikes and protects neurons against excitotoxicity, particularly in cultured rat hippocampal neurons, presumably due to both direct inhibition of presynaptic glutamate release and postsynaptic glutamate receptor-mediated [Ca(2+)](i) signaling. In addition to an indirect inhibitory effect via 5-HT levels, these data suggest a new, possibly direct inhibitory action of fluoxetine on synaptically induced [Ca(2+)](i) spikes and neuronal cell death.

Proanthocyanidin is a phenolic compound present in plants, that has antioxidant, antinociceptive, anti-emetic, and neuroprotective properties. We investigated the actions of proanthocyanidin from grape seeds on 5-hydroxytryptamine (5-HT)(3) receptors in NCB-20 neuroblastoma cells using a whole-cell voltage clamp technique. Co-treatment of proanthocyanidin (0.3-100 µg/ml) and 3 µM 5-HT (near EC(50)) produced a slight inhibition of 5-HT-induced inward peak current (I(5-HT)) in NCB-20 cells, but pretreatment with proanthocyanidin for 30 s before application of 5-HT induced a much larger inhibition of I(5-HT) in an irreversible, concentration- and time-dependent manner (IC(50)=6.5±0.4 µg/ml, Hill coefficient=2.5±0.1). Proanthocyanidin also produced a concentration-dependent inhibition of currents induced by 30 µM 5-HT, near-maximal concentration (IC(50)=22.1±0.4 µg/ml, Hill coefficient=2.4±0.1). High concentrations (≧30 µg/ml) of proanthocyanidin caused a concentration-dependent inhibition of the activation and desensitization of currents induced by 30 µM 5-HT. Further studies showed that pretreatment of 20 µg/ml proanthocyanidin caused not only a rightward shift of the dose-response curve for 5-HT (EC(50) shift from 2.7±0.4 to 6.2±0.5 µM), but also a decreased E(max) (inhibition by 37.5±1.3%). The proanthocyanidin-induced inhibition of 5-HT(3) receptors did not show a significant difference within the testing holding potential ranges (-50-+30 mV). These results suggest that proanthocyanidin inhibits 5-HT(3) receptor function in NCB-20 cells in a noncompetitive mode, and that this inhibitory effect of proanthocyanidin probably contributes to the pharmacological actions of proanthocyanidin.

We examined the effect of 5-hydroxyindole (5-HI) on the potentiation of 5-hydroytryptamine (5-HT)(3) receptor function by ethanol (EtOH) so as to study whether EtOH potentiates channel function through increasing activation or blocking desensitization. We measured 5-HT(3) receptor current using a whole-cell voltage clamp technique with a method of rapid drug application in NCB-20 neuroblastoma cells. The 5-HI, an agent that block receptor desensitization, increased the decay time constant (tau), not the peak of 5-HT(3) receptor-mediated currents induced by 10 microM 5-HT. EtOH did not change the peak amplitude and tau of the current induced by 10 microM 5-HT. Coapplication of EtOH and 5-HI with 5-HT caused no increase in the peak currents, but tau was further increased. Therefore, a further block in desensitization could be induced by 5-HI, despite the presence of EtOH. These results indicate that EtOH potentiates 5-HT(3) receptor function, with these effects due at least in part by increasing channel activation rather than by blocking desensitization.