Toward robust quantification of dopamine and serotonin in mixtures using nano-graphitic carbon sensors.

Monitoring the coordinated signaling of dopamine (DA) and serotonin (5-HT) is important for advancing our understanding of the brain. However, the co-detection and robust quantification of these signals at low concentrations is yet to be demonstrated. Here, we present the quantification of DA and 5-HT using nano-graphitic (NG) sensors together with fast-scan cyclic voltammetry (FSCV) employing an engineered N-shape potential waveform. Our method yields 6% error in quantifying DA and 5-HT analytes present in in vitro mixtures at concentrations below 100 nM. This advance is due to the electrochemical properties of NG sensors which, in combination with the engineered FSCV waveform, provided distinguishable cyclic voltammograms (CVs) for DA and 5-HT. We also demonstrate the generalizability of the prediction model across different NG sensors, which arises from the consistent voltammetric fingerprints produced by our NG sensors. Curiously, the proposed engineered waveform also improves the distinguishability of DA and 5-HT CVs obtained from traditional carbon fiber (CF) microelectrodes. Nevertheless, this improved distinguishability of CVs obtained from CF is inferior to that of NG sensors, arising from differences in the electrochemical properties of the sensor materials. Our findings demonstrate the potential of NG sensors and our proposed FSCV waveform for future brain studies.

Leptin promotes striatal dopamine release via cholinergic interneurons and regionally distinct signaling pathways.

Dopamine (DA) is a critical regulator of striatal network activity and is essential for motor activation and reward-associated behaviors. Previous work has shown that DA is influenced by the reward value of food, as well as by hormonal factors implicated in the regulation of food intake and energy expenditure. Changes in striatal DA signaling also have been linked to aberrant eating patterns. Here we test the effect of leptin, an adipocyte-derived hormone involved in feeding and energy homeostasis regulation, on striatal DA release and uptake. Immunohistochemical evaluation identified leptin receptor expression throughout mouse striatum, including on striatal cholinergic interneurons and their extensive processes. Using fast-scan cyclic voltammetry, we found that leptin causes a concentration-dependent increase in evoked extracellular DA concentration ([DA]o) in dorsal striatum and nucleus accumbens (NAc) core and shell in male mouse striatal slices, and also an increase in the rate of DA uptake. Further, we found that leptin increases cholinergic interneuron excitability, and that the enhancing effect of leptin on evoked [DA]o is lost when nicotinic acetylcholine (ACh) receptors are antagonized or when examined in striatal slices from mice lacking ACh synthesis. Evaluation of signaling pathways underlying leptin's action revealed a requirement for intracellular Ca2+, and the involvement of different downstream pathways in dorsal striatum and NAc core versus NAc shell. These results provide the first evidence for dynamic regulation of DA release and uptake by leptin within brain motor and reward pathways, and highlight the involvement of cholinergic interneurons in this process.SIGNIFICANCE STATEMENTGiven the importance of striatal dopamine in reward, motivation, motor behavior and food intake, identifying the actions of metabolic hormones on dopamine release in striatal subregions should provide new insight into factors that influence dopamine-dependent motivated behaviors. We find that one of these hormones, leptin, boosts striatal dopamine release through a process involving striatal cholinergic interneurons and nicotinic acetylcholine receptors. Moreover, we find that the intracellular cascades downstream from leptin receptor activation underlying enhanced dopamine release differ among striatal subregions. Thus, we not only show that leptin regulates dopamine release, but also identify characteristics of this process that could be harnessed to alter pathological eating behaviors.

Synaptotagmins 1 and 7 Play Complementary Roles in Somatodendritic Dopamine Release.

The molecular mechanisms underlying somatodendritic dopamine (DA) release remain unresolved, despite the passing of decades since its discovery. Our previous work showed robust release of somatodendritic DA in submillimolar extracellular Ca2+ concentration ([Ca2+]o). Here we tested the hypothesis that the high-affinity Ca2+ sensor synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its Ca2+ dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices from male and female mice to monitor evoked DA-dependent D2 receptor-mediated inhibitory currents (D2ICs). Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased pulse train-evoked D2ICs, revealing a functional role for Syt7. The assessment of the Ca2+ dependence of pulse train-evoked D2ICs confirmed robust DA release in submillimolar [Ca2+]o in wild-type (WT) neurons, but loss of this sensitivity with intracellular Syt7 Ab or in Syt7 knock-out (KO) mice. In millimolar [Ca2+]o, pulse train-evoked D2ICs in Syt7 KOs showed a greater reduction in decreased [Ca2+]o than seen in WT mice; the effect on single pulse-evoked DA release, however, did not differ between genotypes. Single-cell application of a Syt1 Ab had no effect on train-evoked D2ICs in WT SNc DA neurons, but did cause a decrease in D2IC amplitude in Syt7 KOs, indicating a functional substitution of Syt1 for Syt7. In addition, Syt1 Ab decreased single pulse-evoked D2ICs in WT cells, indicating the involvement of Syt1 in tonic DA release. Thus, Syt7 and Syt1 play complementary roles in somatodendritic DA release from SNc DA neurons.SIGNIFICANCE STATEMENT The respective Ca2+ dependence of somatodendritic and axonal dopamine (DA) release differs, resulting in the persistence of somatodendritic DA release in submillimolar Ca2+ concentrations too low to support axonal release. We demonstrate that synaptotagmin7 (Syt7), a high-affinity Ca2+ sensor, underlies phasic somatodendritic DA release and its Ca2+ sensitivity in the substantia nigra pars compacta. In contrast, we found that synaptotagmin 1 (Syt1), the Ca2+ sensor underlying axonal DA release, plays a role in tonic, but not phasic, somatodendritic DA release in wild-type mice. However, Syt1 can facilitate phasic DA release after Syt7 deletion. Thus, we show that both Syt1 and Syt7 act as Ca2+ sensors subserving different aspects of somatodendritic DA release processes.

Voluntary Exercise Boosts Striatal Dopamine Release: Evidence for the Necessary and Sufficient Role of BDNF.

Physical exercise improves motor performance in individuals with Parkinson's disease and elevates mood in those with depression. Although underlying factors have not been identified, clues arise from previous studies showing a link between cognitive benefits of exercise and increases in brain-derived neurotrophic factor (BDNF). Here, we investigated the influence of voluntary wheel-running exercise on BDNF levels in the striatum of young male wild-type (WT) mice, and on the striatal release of a key motor-system transmitter, dopamine (DA). Mice were allowed unlimited access to a freely rotating wheel (runners) or a locked wheel (controls) for 30 d. Electrically evoked DA release was quantified in ex vivo corticostriatal slices from these animals using fast-scan cyclic voltammetry. We found that exercise increased BDNF levels in dorsal striatum (dStr) and increased DA release in dStr and in nucleus accumbens core and shell. Increased DA release was independent of striatal acetylcholine (ACh), and persisted after a week of rest. We tested a role for BDNF in the influence of exercise on DA release using mice that were heterozygous for BDNF deletion (BDNF+/-). In contrast to WT mice, evoked DA release did not differ between BDNF+/- runners and controls. Complementary pharmacological studies using a tropomyosin receptor kinase B (TrkB) agonist in WT mouse slices showed that TrkB receptor activation also increased evoked DA release throughout striatum in an ACh-independent manner. Together, these data support a causal role for BDNF in exercise-enhanced striatal DA release and provide mechanistic insight into the beneficial effects of exercise in neuropsychiatric disorders, including Parkinson's, depression, and anxiety.SIGNIFICANCE STATEMENT Exercise has been shown to improve movement and cognition in humans and rodents. Here, we report that voluntary exercise for 30 d leads to an increase in evoked DA release throughout the striatum and an increase in BDNF in the dorsal (motor) striatum. The increase in DA release appears to require BDNF, indicated by the absence of DA release enhancement with running in BDNF+/- mice. Activation of BDNF receptors using a pharmacological agonist was also shown to boost DA release. Together, these data support a necessary and sufficient role for BDNF in exercise-enhanced DA release and provide mechanistic insight into the reported benefits of exercise in individuals with dopamine-linked neuropsychiatric disorders, including Parkinson's disease and depression.

Cell-type-specific disruption of PERK-eIF2α signaling in dopaminergic neurons alters motor and cognitive function.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) has been shown to activate the eIF2α kinase PERK to directly regulate translation initiation. Tight control of PERK-eIF2α signaling has been shown to be necessary for normal long-lasting synaptic plasticity and cognitive function, including memory. In contrast, chronic activation of PERK-eIF2α signaling has been shown to contribute to pathophysiology, including memory impairments, associated with multiple neurological diseases, making this pathway an attractive therapeutic target. Herein, using multiple genetic approaches we show that selective deletion of the PERK in mouse midbrain dopaminergic (DA) neurons results in multiple cognitive and motor phenotypes. Conditional expression of phospho-mutant eIF2α in DA neurons recapitulated the phenotypes caused by deletion of PERK, consistent with a causal role of decreased eIF2α phosphorylation for these phenotypes. In addition, deletion of PERK in DA neurons resulted in altered de novo translation, as well as changes in axonal DA release and uptake in the striatum that mirror the pattern of motor changes observed. Taken together, our findings show that proper regulation of PERK-eIF2α signaling in DA neurons is required for normal cognitive and motor function in a non-pathological state, and also provide new insight concerning the onset of neuropsychiatric disorders that accompany UPR failure.

Interactions between Soluble Species of ß-Amyloid and α-Synuclein Promote Oligomerization while Inhibiting Fibrillization.

Aggregations of ß-amyloid (Aß) and α-synuclein (αS) into oligomeric and fibrillar assemblies are the pathological hallmarks of Alzheimer's and Parkinson's diseases, respectively. Although Aß and αS affect different regions of the brain and are separated at the cellular level, there is evidence of their eventual interaction in the pathology of both disorders. Characterization of interactions of Aß and αS at various stages of their aggregation pathways could reveal mechanisms and therapeutic targets for the prevention and cure of these neurodegenerative diseases. In this study, we comprehensively examined the interactions and their molecular manifestations using an array of characterization tools. We show for the first time that αS monomers and oligomers, but not αS fibrils, inhibit Aß fibrillization while promoting oligomerization of Aß monomers and stabilizing preformed Aß oligomers via coassembly, as judged by Thioflavin T fluorescence, transmission electron microscopy, and SDS- and native-PAGE with fluorescently labeled peptides/proteins. In contrast, soluble Aß species, such as monomers and oligomers, aggregate into fibrils, when incubated alone under the otherwise same condition. Our study provides evidence that the interactions with αS soluble species, responsible for the effects, are mediated primarily by the C-terminus of Aß, when judged by competitive immunoassays using antibodies recognizing various fragments of Aß. We also show that the C-terminus of Aß is a primary site for its interaction with αS fibrils. Collectively, these data demonstrate aggregation state-specific interactions between αS and Aß and offer insight into a molecular basis of synergistic biological effects between the two polypeptides.

Interactions between insulin and diet on striatal dopamine uptake kinetics in rodent brain slices.

Diet influences dopamine transmission in motor- and reward-related basal ganglia circuitry. In part, this reflects diet-dependent regulation of circulating and brain insulin levels. Activation of striatal insulin receptors amplifies axonal dopamine release in brain slices, and regulates food preference in vivo. The effect of insulin on dopamine release is indirect, and requires striatal cholinergic interneurons that express insulin receptors. However, insulin also acts directly on dopamine axons to increase dopamine uptake by promoting dopamine transporter (DAT) surface expression, counteracting enhanced dopamine release. Here, we determined the functional consequences of acute insulin exposure and chronic diet-induced changes in insulin on DAT activity after evoked dopamine release in striatal slices from adult ad-libitum fed (AL) rats and mice, and food-restricted (FR) or high-fat/high-sugar obesogenic (OB) diet rats. Uptake kinetics were assessed by fitting evoked dopamine transients to the Michaelis-Menten equation and extracting Cpeak and Vmax . Insulin (30 nm) increased both parameters in the caudate putamen and nucleus accumbens core of AL rats in an insulin receptor- and PI3-kinase-dependent manner. A pure effect of insulin on uptake was unmasked using mice lacking striatal acetylcholine, in which increased Vmax caused a decrease in Cpeak . Diet also influenced Vmax , which was lower in FR vs. AL. The effects of insulin on Cpeak and Vmax were amplified by FR but blunted by OB, consistent with opposite consequences of these diets on insulin levels and insulin receptor sensitivity. Overall, these data reveal acute and chronic effects of insulin and diet on dopamine release and uptake that will influence brain reward pathways.

Inhibitory and excitatory neuromodulation by hydrogen peroxide: translating energetics to information.

Historically, brain neurochemicals have been broadly classified as energetic or informational. However, increasing evidence implicates metabolic substrates and byproducts as signalling agents, which blurs the boundary between energy and information, and suggests the introduction of a new category for 'translational' substances that convey changes in energy state to information. One intriguing example is hydrogen peroxide (H2 O2 ), which is a small, readily diffusible molecule. Produced during mitochondrial respiration, this reactive oxygen species, can mediate dynamic regulation of neuronal activity and transmitter release by activating inhibitory ATP-sensitive K(+) (KATP ) channels, as well as a class of excitatory non-selective cation channels, TRPM2. Studies using ex vivo guinea pig brain slices have revealed that activity-generated H2 O2 can act via KATP channels to inhibit dopamine release in dorsal striatum and dopamine neuron activity in the substantia nigra pars compacta. In sharp contrast, endogenously generated H2 O2 enhances the excitability of GABAergic projection neurons in the dorsal striatum and substantia nigra pars reticulata by activating TRPM2 channels. These studies suggest that the balance of excitation vs. inhibition produced in a given cell by metabolically generated H2 O2 will be dictated by the relative abundance of H2 O2 -sensitive ion channel targets that receive this translational signal.

TRPM2 channels are required for NMDA-induced burst firing and contribute to H(2)O(2)-dependent modulation in substantia nigra pars reticulata GABAergic neurons.

Substantia nigra pars reticulata (SNr) GABAergic neurons are projection neurons that convey output from the basal ganglia to target structures. These neurons exhibit spontaneous regular firing, but also exhibit burst firing in the presence of NMDA or when excitatory glutamatergic input to the SNr is activated. Notably, an increase in burst firing is also seen in Parkinson's disease. Therefore, elucidating conductances that mediate spontaneous activity and changes of firing pattern in these neurons is essential for understanding how the basal ganglia control movement. Using ex vivo slices of guinea pig midbrain, we show that SNr GABAergic neurons express transient receptor potential melastatin 2 (TRPM2) channels that underlie NMDA-induced burst firing. Furthermore, we show that spontaneous firing rate and burst activity are modulated by the reactive oxygen species H(2)O(2) acting via TRPM2 channels. Thus, our results indicate that activation of TRPM2 channels is necessary for burst firing in SNr GABAergic neurons and their responsiveness to modulatory H(2)O(2). These findings have implications not only for normal regulation, but also for Parkinson's disease, which involves excitotoxicity and oxidative stress.

GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABAA receptors.

Striatal dopamine axons co-release dopamine and gamma-aminobutyric acid (GABA), using GABA provided by uptake via GABA transporter-1 (GAT1). Functions of GABA co-release are poorly understood. We asked whether co-released GABA autoinhibits dopamine release via axonal GABA type A receptors (GABAARs), complementing established inhibition by dopamine acting at axonal D2 autoreceptors. We show that dopamine axons express α3-GABAAR subunits in mouse striatum. Enhanced dopamine release evoked by single-pulse optical stimulation in striatal slices with GABAAR antagonism confirms that an endogenous GABA tone limits dopamine release. Strikingly, an additional inhibitory component is seen when multiple pulses are used to mimic phasic axonal activity, revealing the role of GABAAR-mediated autoinhibition of dopamine release. This autoregulation is lost in conditional GAT1-knockout mice lacking GABA co-release. Given the faster kinetics of ionotropic GABAARs than G-protein-coupled D2 autoreceptors, our data reveal a mechanism whereby co-released GABA acts as a first responder to dampen phasic-to-tonic dopamine signaling.

Actions and Consequences of Insulin in the Striatum.

Insulin crosses the blood-brain barrier to enter the brain from the periphery. In the brain, insulin has well-established actions in the hypothalamus, as well as at the level of mesolimbic dopamine neurons in the midbrain. Notably, insulin also acts in the striatum, which shows abundant expression of insulin receptors (InsRs) throughout. These receptors are found on interneurons and striatal projections neurons, as well as on glial cells and dopamine axons. A striking functional consequence of insulin elevation in the striatum is promoting an increase in stimulated dopamine release. This boosting of dopamine release involves InsRs on cholinergic interneurons, and requires activation of nicotinic acetylcholine receptors on dopamine axons. Opposing this dopamine-enhancing effect, insulin also increases dopamine uptake through the action of insulin at InsRs on dopamine axons. Insulin acts on other striatal cells as well, including striatal projection neurons and astrocytes that also influence dopaminergic transmission and striatal function. Linking these cellular findings to behavior, striatal insulin signaling is required for the development of flavor-nutrient learning, implicating insulin as a reward signal in the brain. In this review, we discuss these and other actions of insulin in the striatum, including how they are influenced by diet and other physiological states.

Enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial Parkinson's disease mutation G2019S.

PARK8/LRRK2 (leucine-rich repeat kinase 2) was recently identified as a causative gene for autosomal dominant Parkinson's disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events.

Glutamatergic signaling by mesolimbic dopamine neurons in the nucleus accumbens.

Recent evidence suggests the intriguing possibility that midbrain dopaminergic (DAergic) neurons may use fast glutamatergic transmission to communicate with their postsynaptic targets. Because of technical limitations, direct demonstration of the existence of this signaling mechanism has been limited to experiments using cell culture preparations that often alter neuronal function including neurotransmitter phenotype. Consequently, it remains uncertain whether glutamatergic signaling between DAergic neurons and their postsynaptic targets exists under physiological conditions. Here, using an optogenetic approach, we provide the first conclusive demonstration that mesolimbic DAergic neurons in mice release glutamate and elicit excitatory postsynaptic responses in projection neurons of the nucleus accumbens. In addition, we describe the properties of the postsynaptic glutamatergic responses of these neurons during experimentally evoked burst firing of DAergic axons that reproduce the reward-related phasic population activity of the mesolimbic projection. These observations indicate that, in addition to DAergic mechanisms, mesolimbic reward signaling may involve glutamatergic transmission.

Subsecond regulation of striatal dopamine release by pre-synaptic KATP channels.

ATP-sensitive K(+) (K(ATP)) channels are composed of pore-forming subunits, typically Kir6.2 in neurons, and regulatory sulfonylurea receptor subunits. In dorsal striatum, activity-dependent H(2)O(2) produced from glutamate receptor activation inhibits dopamine release via K(ATP) channels. Sources of modulatory H(2)O(2) include striatal medium spiny neurons, but not dopaminergic axons. Using fast-scan cyclic voltammetry in guinea-pig striatal slices and immunohistochemistry, we determined the time window for H(2)O(2)/K(ATP)-channel-mediated inhibition and assessed whether modulatory K(ATP) channels are on dopaminergic axons. Comparison of paired-pulse suppression of dopamine release in the absence and presence of glibenclamide, a K(ATP)-channel blocker, or mercaptosuccinate, a glutathione peroxidase inhibitor that enhances endogenous H(2)O(2) levels, revealed a time window for inhibition of 500-1000 ms after stimulation. Immunohistochemistry demonstrated localization of Kir6.2 K(ATP)-channel subunits on dopaminergic axons. Consistent with the presence of functional K(ATP) channels on dopaminergic axons, K(ATP)-channel openers, diazoxide and cromakalim, suppressed single-pulse evoked dopamine release. Although cholinergic interneurons that tonically regulate dopamine release also express K(ATP) channels, diazoxide did not induce the enhanced frequency responsiveness of dopamine release seen with nicotinic-receptor blockade. Together, these studies reveal subsecond regulation of striatal dopamine release by endogenous H(2)O(2) acting at K(ATP) channels on dopaminergic axons, including a role in paired-pulse suppression.

SKF-83566, a D1-dopamine receptor antagonist, inhibits the dopamine transporter.

Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1)-receptor activation in axonal DA release regulation in dorsal striatum using a D(1)-receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of ∼ 65% in 5 µM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 µM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3)H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 µM. Binding studies with [(3)H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 µM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT.

Activity-dependent somatodendritic dopamine release in the substantia nigra autoinhibits the releasing neuron.

Somatodendritic dopamine (DA) release from midbrain DA neurons activates D2 autoreceptors on these cells to regulate their activity. However, the source of autoregulatory DA remains controversial. Here, we test the hypothesis that D2 autoreceptors on a given DA neuron in the substantia nigra pars compacta (SNc) are activated primarily by DA released from that same cell, rather than from its neighbors. Voltage-clamp recording allows monitoring of evoked D2-receptor-mediated inhibitory currents (D2ICs) in SNc DA neurons as an index of DA release. Single-cell application of antibodies to Na+ channels via the recording pipette decreases spontaneous activity of recorded neurons and attenuates evoked D2ICs; antibodies to SNAP-25, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, also decrease D2IC amplitude. Evoked D2ICs are nearly abolished by the light chain of botulinum neurotoxin A, which cleaves SNAP-25, whereas synaptically activated GABAB-receptor-mediated currents are unaffected. Thus, somatodendritic DA release in the SNc autoinhibits the neuron that releases it.

Mobilization of calcium from intracellular stores facilitates somatodendritic dopamine release.

Somatodendritic dopamine (DA) release in the substantia nigra pars compacta (SNc) shows a limited dependence on extracellular calcium concentration ([Ca(2+)](o)), suggesting the involvement of intracellular Ca(2+) stores. Here, using immunocytochemistry we demonstrate the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) that sequesters cytosolic Ca(2+) into the endoplasmic reticulum (ER), as well as inositol 1,4,5-triphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in DAergic neurons. Notably, RyRs were clustered at the plasma membrane, poised for activation by Ca(2+) entry. Using fast-scan cyclic voltammetry to monitor evoked extracellular DA concentration ([DA](o)) in midbrain slices, we found that SERCA inhibition by cyclopiazonic acid (CPA) decreased evoked [DA](o) in the SNc, indicating a functional role for ER Ca(2+) stores in somatodendritic DA release. Implicating IP(3)R-dependent stores, an IP(3)R antagonist, 2-APB, also decreased evoked [DA](o). Moreover, DHPG, an agonist of group I metabotropic glutamate receptors (mGluR1s, which couple to IP(3) production), increased somatodendritic DA release, whereas CPCCOEt, an mGluR1 antagonist, suppressed it. Release suppression by mGluR1 blockade was prevented by 2-APB or CPA, indicating facilitation of DA release by endogenous glutamate acting via mGluR1s and IP(3)R-gated Ca(2+) stores. Similarly, activation of RyRs by caffeine increased [Ca(2+)](i) and elevated evoked [DA](o). The increase in DA release was prevented by a RyR blocker, dantrolene, and by CPA. Importantly, the efficacy of dantrolene was enhanced in low [Ca(2+)](o), suggesting a mechanism for maintenance of somatodendritic DA release with limited Ca(2+) entry. Thus, both mGluR1-linked IP(3)R- and RyR-dependent ER Ca(2+) stores facilitate somatodendritic DA release in the SNc.

Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling.

Hydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H(2)O(2) signaling, which activates ATP-sensitive potassium (K(ATP)) channels to inhibit dopamine release. However, the origin of this modulatory H(2)O(2) has been elusive. Here we addressed three possible sources of H(2)O(2) produced for rapid neuronal signaling in striatum: mitochondrial respiration, monoamine oxidase (MAO), and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H(2)O(2) and tissue analysis of ATP, we found that coapplication of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H(2)O(2) production, but maintained tissue ATP content. Strikingly, coapplication of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H(2)O(2) in release modulation. In contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H(2)O(2) production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H(2)O(2) generation for dynamic neuronal signaling.

Dysregulation of striatal dopamine release in a mouse model of dystonia.

Dystonia is a neurological disorder characterized by involuntary movements. We examined striatal dopamine (DA) function in hyperactive transgenic (Tg) mice generated as a model of dystonia. Evoked extracellular DA concentration was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry in striatal slices from non-Tg mice, Tg mice with a positive motor phenotype, and phenotype-negative Tg littermates. Peak single-pulse evoked extracellular DA concentration was significantly lower in phenotype-positive mice than in non-Tg or phenotype-negative mice, but indistinguishable between non-Tg and phenotype-negative mice. Phenotype-positive mice also had higher functional D2 DA autoreceptor sensitivity than non-Tg mice, which would be consistent with lower extracellular DA concentration in vivo. Multiple-pulse (phasic) stimulation (five pulses, 10-100 Hz) revealed an enhanced frequency dependence of evoked DA release in phenotype-positive versus non-Tg or phenotype-negative mice, which was exacerbated when extracellular Ca(2+) concentration was lowered. Enhanced sensitivity to phasic stimulation in phenotype-positive mice was reminiscent of the pattern seen with antagonism of nicotinic acetylcholine receptors. Consistent with a role for altered cholinergic regulation, the difference in phasic responsiveness among groups was lost when nicotinic receptors were blocked by mecamylamine. Together, these data implicate compromised DA release regulation, possibly from cholinergic dysfunction, in the motor symptoms of this dystonia model.