Dopamine Release Neuroenergetics in Mouse Striatal Slices.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Dopamine (DA) neurons in the substantia nigra pars compacta, which have axonal projections to the dorsal striatum (dSTR), degenerate in PD. In contrast, DA neurons in the ventral tegmental area, with axonal projections to the ventral striatum, including the nucleus accumbens (NAcc) shell, are largely spared. This study aims to uncover the relative contributions of glycolysis and oxidative phosphorylation (OxPhos) to DA release in the striatum. We measured evoked DA release in mouse striatal brain slices using fast-scan cyclic voltammetry applied every two minutes. Blocking OxPhos resulted in a greater reduction in evoked DA release in the dSTR when compared to the NAcc shell, while blocking glycolysis caused a more significant decrease in evoked DA release in the NAcc shell than in the dSTR. Furthermore, when glycolysis was bypassed in favor of direct OxPhos, evoked DA release in the NAcc shell decreased by approximately 50% over 40 min, whereas evoked DA release in the dSTR was largely unaffected. These results demonstrate that the dSTR relies primarily on OxPhos for energy production to maintain evoked DA release, whereas the NAcc shell depends more on glycolysis. Consistently, two-photon imaging revealed higher oxidation levels of DA terminals in the dSTR than in the NAcc shell. Together, these findings partly explain the selective vulnerability of DA terminals in the dSTR to degeneration in PD.

Apoptosis signal-regulating kinase 1 (Ask1) deficiency alleviates MPP+-induced impairment of evoked dopamine release in the mouse hippocampus.

The dopaminergic system is susceptible to dysfunction in numerous neurological diseases, including Parkinson's disease (PD). In addition to motor symptoms, some PD patients may experience non-motor symptoms, including cognitive and memory deficits. A possible explanation for their manifestation is a disturbed pattern of dopamine release in brain regions involved in learning and memory, such as the hippocampus. Therefore, investigating neuropathological alterations in dopamine release prior to neurodegeneration is imperative. This study aimed to characterize evoked hippocampal dopamine release and assess the impact of the neurotoxin MPP+ using a genetically encoded dopamine sensor and gene expression analysis. Additionally, considering the potential neuroprotective attributes demonstrated by apoptosis signal-regulating kinase 1 (Ask1) in various animal-disease-like models, the study also aimed to determine whether Ask1 knockdown restores MPP+-altered dopamine release in acute hippocampal slices. We applied variations of low- and high-frequency stimulation to evoke dopamine release within different hippocampal regions and discovered that acute application of MPP+ reduced the amount of dopamine released and hindered the recovery of dopamine release after repeated stimulation. In addition, we observed that Ask1 deficiency attenuated the detrimental effects of MPP+ on the recovery of dopamine release after repeated stimulation. RNA sequencing analysis indicated that genes associated with the synaptic pathways are involved in response to MPP+ exposure. Notably, Ask1 deficiency was found to downregulate the expression of Slc5a7, a gene encoding a sodium-dependent high-affinity choline transporter that regulates acetylcholine levels. Respective follow-up experiments indicated that Slc5a7 plays a role in Ask1 deficiency-mediated protection against MPP+ neurotoxicity. In addition, increasing acetylcholine levels using an acetylcholinesterase inhibitor could exacerbate the toxicity of MPP+. In conclusion, our data imply that the modulation of the dopamine-acetylcholine balance may be a crucial mechanism of action underlying the neuroprotective effects of Ask1 deficiency in PD.

Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine co-varies with risk factors for Parkinson's.

Ca2+ entry into nigrostriatal dopamine (DA) neurons and axons via L-type voltage-gated Ca2+ channels (LTCCs) contributes, respectively, to pacemaker activity and DA release and has long been thought to contribute to vulnerability to degeneration in Parkinson's disease. LTCC function is greater in DA axons and neurons from substantia nigra pars compacta than from ventral tegmental area, but this is not explained by channel expression level. We tested the hypothesis that LTCC control of DA release is governed rather by local mechanisms, focussing on candidate biological factors known to operate differently between types of DA neurons and/or be associated with their differing vulnerability to parkinsonism, including biological sex, α-synuclein, DA transporters (DATs) and calbindin-D28k (Calb1). We detected evoked DA release ex vivo in mouse striatal slices using fast-scan cyclic voltammetry and assessed LTCC support of DA release by detecting the inhibition of DA release by the LTCC inhibitors isradipine or CP8. Using genetic knockouts or pharmacological manipulations, we identified that striatal LTCC support of DA release depended on multiple intersecting factors, in a regionally and sexually divergent manner. LTCC function was promoted by factors associated with Parkinsonian risk, including male sex, α-synuclein, DAT and a dorsolateral co-ordinate, but limited by factors associated with protection, that is, female sex, glucocerebrosidase activity, Calb1 and ventromedial co-ordinate. Together, these data show that LTCC function in DA axons and isradipine effect are locally governed and suggest they vary in a manner that in turn might impact on, or reflect, the cellular stress that leads to parkinsonian degeneration.

Ultrastructural analysis of nigrostriatal dopaminergic terminals in a knockin mouse model of DYT1 dystonia.

DYT1 dystonia is associated with decreased striatal dopamine release. In this study, we examined the possibility that ultrastructural changes of nigrostriatal dopamine terminals could contribute to this neurochemical imbalance using a serial block face/scanning electron microscope (SBF/SEM) and three-dimensional reconstruction to analyse striatal tyrosine hydroxylase-immunoreactive (TH-IR) terminals and their synapses in a DYT1(ΔE) knockin (DYT1-KI) mouse model of DYT1 dystonia. Furthermore, to study possible changes in vesicle packaging capacity of dopamine, we used transmission electron microscopy to assess the synaptic vesicle size in striatal dopamine terminals. Quantitative comparative analysis of 80 fully reconstructed TH-IR terminals in the WT and DYT1-KI mice indicate (1) no significant difference in the volume of TH-IR terminals; (2) no major change in the proportion of axo-spinous versus axo-dendritic synapses; (3) no significant change in the post-synaptic density (PSD) area of axo-dendritic synapses, while the PSDs of axo-spinous synapses were significantly smaller in DYT1-KI mice; (4) no significant change in the contact area between TH-IR terminals and dendritic shafts or spines, while the ratio of PSD area/contact area decreased significantly for both axo-dendritic and axo-spinous synapses in DYT1-KI mice; (5) no significant difference in the mitochondria volume; and (6) no significant difference in the synaptic vesicle area between the two groups. Altogether, these findings suggest that abnormal morphometric changes of nigrostriatal dopamine terminals and their post-synaptic targets are unlikely to be a major source of reduced striatal dopamine release in DYT1 dystonia.

Human Embryonic Stem Cell-Derived Immature Midbrain Dopaminergic Neurons Transplanted in Parkinsonian Monkeys.

Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DANs), and Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson's disease (PD) patients. Here, we obtained well-characterized DANs from hESCs and transplanted them into two parkinsonian monkeys to assess their behavioral and imaging changes. DANs from hESCs expressed dopaminergic markers, generated action potentials, and released dopamine (DA) in vitro. These neurons were transplanted bilaterally into the putamen of parkinsonian NHPs, and using magnetic resonance imaging techniques, we calculated the fractional anisotropy (FA) and mean diffusivity (MD), both employed for the first time for these purposes, to detect in vivo axonal and cellular density changes in the brain. Likewise, positron-emission tomography scans were performed to evaluate grafted DANs. Histological analyses identified grafted DANs, which were quantified stereologically. After grafting, animals showed signs of partially improved motor behavior in some of the HALLWAY motor tasks. Improvement in motor evaluations was inversely correlated with increases in bilateral FA. MD did not correlate with behavior but presented a negative correlation with FA. We also found higher 11C-DTBZ binding in positron-emission tomography scans associated with grafts. Higher DA levels measured by microdialysis after stimulation with a high-potassium solution or amphetamine were present in grafted animals after ten months, which has not been previously reported. Postmortem analysis of NHP brains showed that transplanted DANs survived in the putamen long-term, without developing tumors, in immunosuppressed animals. Although these results need to be confirmed with larger groups of NHPs, our molecular, behavioral, biochemical, and imaging findings support the integration and survival of human DANs in this pre-clinical PD model.

Role of voltage-dependent calcium channels on the striatal in vivo dopamine release induced by the organophosphorus pesticide glyphosate.

In the present study, we investigated the role of voltage-sensitive calcium channels (VSCCs) on the striatal dopamine release induced by the pesticide glyphosate (GLY) using selective VSCC inhibitors. The dopamine levels were measured by in vivo cerebral microdialysis coupled to HPLC-ED. Nicardipine (L-type VSCC antagonist) or ω-conotoxin MVIIC (non-selective P/Q-type antagonist) had no effect on dopamine release induced by 5 mM GLY. In contrast, flunarizine (T-type antagonist) or ω-conotoxin GVIA (neuronal N-type antagonist) significantly reduced GLY-stimulated dopamine release. These results suggest that GLY-induced dopamine release depends on extracellular calcium and its influx through the T- and N-type VSCCs. These findings were corroborated by molecular docking, which allowed us to establish a correlation between the effect of GLY on blocked VSCC with the observed dopamine release. We propose new molecular targets of GLY in the dorsal striatum, which could have important implications for the assessment of pesticide risks in non-target organisms.

Striking Neurochemical and Behavioral Differences in the Mode of Action of Selegiline and Rasagiline.

Selegiline and rasagiline are two selective monoamine oxidase B (MAO-B) inhibitors used in the treatment of Parkinson's disease. In their clinical application, however, differences in L-dopa-sparing potencies have been observed. The aim of this study was to find neurochemical and behavioral explanations for the antiparkinsonian effects of these drugs. We found that selegiline possesses a dopaminergic enhancer effect: it stimulated the electrically induced [3H]dopamine release without influencing the resting [3H]dopamine release from rat striatal slices in 10-10-10-9 mol/L concentrations. Rasagiline added in 10-13 to 10-5 mol/L concentrations did not alter the resting or electrically stimulated [3H]dopamine release. Rasagiline (10-9 mol/L), however, suspended the stimulatory effect of selegiline on the electrically induced [3H]dopamine release. The trace amine-associated receptor 1 (TAAR1) antagonist EPPTB (10-8-10-7 mol/L) also inhibited the stimulatory effect of selegiline on [3H]dopamine release. The effect of selegiline in its enhancer dose (5.33 nmol/kg) against tetrabenazine-induced learning deficit measured in a shuttle box apparatus was abolished by a 5.84 nmol/kg dose of rasagiline. The selegiline metabolite (-)methamphetamine (10-9 mol/L) also exhibited enhancer activity on [3H]dopamine release. We have concluded that selegiline acts as an MAO-B inhibitor and a dopaminergic enhancer drug, and the latter relates to an agonist effect on TAAR1. In contrast, rasagiline is devoid of enhancer activity but may act as an antagonist on TAAR1.

Dopamine Release Impairments Accompany Movement Vigor Deficiency in an Exercise-Induced Fatigue Mouse Model.

Fatigue is a common symptom in neurological diseases with a complex cause, involving the influence of events occurring in both the central and peripheral nervous systems. When people suffer from fatigue, a general decline in their movement performance typically occurs. The neural representation of dopamine signaling in the striatum plays a crucial role in movement regulation. Movement vigor is regulated by dopamine-dependent neuron activity in the striatum. However, whether exercise-induced fatigue alters stimulated dopamine release and further affects movement vigor has not been described. Here, for the first time, we used fast-scan cyclic voltammetry to demonstrate the effect of exercise-induced fatigue on stimulated dopamine release in the striatum in combination with a fiber photometry system to observe the excitability of striatal neurons. The movement vigor of mice was reduced, and after fatigue, the balance of excitability of striatal neurons regulated by dopamine projections was disturbed, which was induced by a reduction in dopamine release. Additionally, D2DR regulation may serve as a targeted intervention to alleviate exercise-induced fatigue and promote fatigue recovery.

Neurotoxic effects of exposure to glyphosate in rat striatum: Effects and mechanisms of action on dopaminergic neurotransmission.

The main objective of this study was to evaluate the effects and possible mechanisms of action of glyphosate and a glyphosate-based herbicide (GBH) on dopaminergic neurotransmission in the rat striatum. Acute exposure to glyphosate or GBH, administered by systemic (75 or 150 mg/kg, i.p.) or intrastriatal (1, 5, or 10 mM for 1 h) routes, produced significant concentration-dependent increases in dopamine release measured in vivo by cerebral microdialysis coupled to HPLC with electrochemical detection. Systemic administration of glyphosate also significantly impaired motor control and decreased striatal acetylcholinesterase activity and antioxidant capacity. At least two mechanisms can be proposed to explain the glyphosate-induced increases in extracellular dopamine levels: increased exocytotic dopamine release from synaptic vesicles or inhibition of dopamine transporter (DAT). Thus, we investigated the effects of intrastriatal administration of glyphosate (5 mM) in animals pretreated with tetrodotoxin (TTX) or reserpine. It was observed that TTX (10 or 20 µM) had no significant effect on glyphosate-induced dopamine release, while reserpine (10 mg/kg i.p) partially but significantly reduced the dopamine release. When glyphosate was coinfused with nomifensine (50 µM), the increase in dopamine levels was significantly higher than that observed with glyphosate or nomifensine alone. So, two possible hypotheses could explain this additive effect: both glyphosate and nomifensine act through different mechanisms at the dopaminergic terminals to increase dopamine levels; or both nomifensine and glyphosate act on DAT, with glyphosate simultaneously inhibiting reuptake and stimulating dopamine release by reversing the DAT function. Future research is needed to determine the effects of this pesticide at environmentally relevant doses.

Impaired dopamine release in Parkinson's disease.

Parkinson's disease is the second most common neurodegenerative disease and yet the early pathophysiological events of the condition and sequences of dysfunction remain unclear. The loss of dopaminergic neurons and reduced levels of striatal dopamine are descriptions used interchangeably as underlying the motor deficits in Parkinson's disease. However, decades of research suggest that dopamine release deficits in Parkinson's disease do not occur only after cell death, but that there is dysfunction or dysregulation of axonal dopamine release before cell loss. Here we review the evidence for dopamine release deficits prior to neurodegeneration in Parkinson's disease, drawn from a large and emerging range of Parkinson's disease models, and the mechanisms by which these release deficits occur. The evidence indicates that impaired dopamine release can result from disruption to a diverse range of Parkinson's disease-associated genetic and molecular disturbances, and can be considered as a potential pathophysiological hallmark of Parkinson's disease.

Dopamine Transmission Imbalance in Neuroinflammation: Perspectives on Long-Term COVID-19.

Dopamine (DA) is a key neurotransmitter in the basal ganglia, implicated in the control of movement and motivation. Alteration of DA levels is central in Parkinson's disease (PD), a common neurodegenerative disorder characterized by motor and non-motor manifestations and deposition of alpha-synuclein (α-syn) aggregates. Previous studies have hypothesized a link between PD and viral infections. Indeed, different cases of parkinsonism have been reported following COVID-19. However, whether SARS-CoV-2 may trigger a neurodegenerative process is still a matter of debate. Interestingly, evidence of brain inflammation has been described in postmortem samples of patients infected by SARS-CoV-2, which suggests immune-mediated mechanisms triggering the neurological sequelae. In this review, we discuss the role of proinflammatory molecules such as cytokines, chemokines, and oxygen reactive species in modulating DA homeostasis. Moreover, we review the existing literature on the possible mechanistic interplay between SARS-CoV-2-mediated neuroinflammation and nigrostriatal DAergic impairment, and the cross-talk with aberrant α-syn metabolism.

Behavioral encoding across timescales by region-specific dopamine dynamics.

The dorsal (DS) and ventral striatum (VS) receive dopaminergic projections that control motor functions and reward-related behavior. It remains poorly understood how dopamine release dynamics across different temporal scales in these regions are coupled to behavioral outcomes. Here, we employ the dopamine sensor dLight1.3b together with multiregion fiber photometry and machine learning-based analysis to decode dopamine dynamics across the striatum during self-paced exploratory behavior in mice. Our data show a striking coordination of rapidly fluctuating signal in the DS, carrying information across dopamine levels, with a slower signal in the VS, consisting mainly of slow-paced transients. Importantly, these release dynamics correlated with discrete behavioral motifs, such as turns, running, and grooming on a subsecond-to-minute time scale. Disruption of dopamine dynamics with cocaine caused randomization of action selection sequencing and disturbance of DS-VS coordination. The data suggest that distinct dopamine dynamics of DS and VS jointly encode behavioral sequences during unconstrained activity with DS modulating the stringing together of actions and VS the signal to initiate and sustain the selected action.

Rate of onset of dopamine transporter inhibitors assessed with intracranial self-stimulation and in vivo dopamine photometry in rats.

Drug self-administration and intracranial self-stimulation (ICSS) are two preclinical behavioral procedures used to predict abuse potential of drugs, and abuse-related drug effects in both procedures are thought to depend on increased mesolimbic dopamine (DA) signaling. Drug self-administration and ICSS yield concordant metrics of abuse potential across a diverse range of drug mechanisms of action. The "rate of onset," defined as the velocity with which a drug produces its effect once administered, has also been implicated as a determinant of abuse-related drug effects in self-administration procedures, but this variable has not been systematically examined in ICSS. Accordingly, this study compared ICSS effects produced in rats by three DA transporter inhibitors that have different rates of onset (fastest to slowest: cocaine, WIN-35428, RTI-31) and that produced progressively weaker metrics of abuse potential in a drug self-administration procedure in rhesus monkeys. Additionally, in vivo photometry using the fluorescent DA sensor dLight1.1 targeted to the nucleus accumbens (NAc) was used to assess the time course of extracellular DA levels as a neurochemical correlate of behavioral effects. All three compounds produced ICSS facilitation and increased DA levels assessed by dLight. In both procedures, the rank order of onset rate was cocaine > WIN-35428 > RTI-31; however, in contrast to monkey drug self-administration results, maximum effects did not differ across compounds. These results provide additional evidence that drug-induced increases in DA drive ICSS facilitation in rats and illustrate the utility of both ICSS and photometry to evaluate the time course and magnitude of abuse-related drug effects in rats.

Central nervous system interaction and crosstalk between nAChRs and other ionotropic and metabotropic neurotransmitter receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in both the peripheral and the central nervous systems. nAChRs exert a crucial modulatory influence on several brain biological processes; they are involved in a variety of neuronal diseases including Parkinson's disease, Alzheimer's disease, epilepsy, and nicotine addiction. The influence of nAChRs on brain function depends on the activity of other neurotransmitter receptors that co-exist with nAChRs on neurons. In fact, the crosstalk between receptors is an important mechanism of neurotransmission modulation and plasticity. This may be due to converging intracellular pathways but also occurs at the membrane level, because of direct physical interactions between receptors. In this line, this review is dedicated to summarizing how nAChRs and other ionotropic and metabotropic receptors interact and the relevance of nAChRs cross-talks in modulating various neuronal processes ranging from the classical modulation of neurotransmitter release to neuron plasticity and neuroprotection.

Pharmacokinetic and Permeation Studies in Rat Brain of Natural Compounds Led to Investigate Eugenol as Direct Activator of Dopamine Release in PC12 Cells.

Eugenol, cinnamaldehyde and D-limonene, the main components of natural essential oils, are endowed with antioxidant and anti-inflammatory properties which allow them to induce beneficial effects on intestinal, cardiac and neuronal levels. In order to characterize their pharmacokinetic profiles and aptitude to permeate in the central nervous system after intravenous and oral administration to rats, new analytical procedures, easily achievable with HPLC-UV techniques, were developed. The terminal half-lives of these compounds range from 12.4 ± 0.9 (D-limonene) and 23.1 ± 1.6 min (cinnamaldehyde); their oral bioavailability appears relatively poor, ranging from 4.25 ± 0.11% (eugenol) to 7.33 ± 0.37% (cinnamaldehyde). Eugenol evidences a marked aptitude to permeate in the cerebrospinal fluid (CSF) of rats following both intravenous and oral administrations, whereas cinnamaldehyde appears able to reach the CSF only after intravenous administration; limonene is totally unable to permeate in the CSF. Eugenol was therefore recruited for in vitro studies of viability and time-/dose-dependent dopamine release in neuronal differentiated PC12 cells (a recognized cellular model mimicking dopaminergic neurons), evidencing its ability to increase cell viability and to induce dopamine release according to a U-shaped time-course curve. Moreover, concentration-response data suggest that eugenol may induce beneficial effects against Parkinson's disease after oral administration.

Role of voltage-sensitive Ca2+ channels in the in vivo dopamine release induced by the organophosphorus pesticide glufosinate ammonium in rat striatum.

The possible role of voltage-sensitive calcium channels (VSCC) activation in the glufosinate ammonium (GLA)-induced dopamine release was investigated using selective VSCC blockers and the dopamine levels were measured by HPLC from samples obtained by in vivo cerebral microdialysis. While pretreatment with 10 µM flunarizine (T-type VSCC antagonist) or nicardipine (L-type VSCC antagonist) had no statistically significant effect on dopamine release induced by 10 mM GLA, pretreatment with 100 µM of both antagonists, or 20 µM ω-conotoxin MVIIC (non-selective P/Q-type VSCC antagonist) significantly decreased the GLA-induced dopamine release over 72.2%, 73%, and 70.2%, respectively. Administration of the specific antagonist of neuronal N-type VSCCs, the ω-conotoxin GVIA (20 µM), produced an almost complete blockade of in vivo dopamine release induced by GLA. These results show that GLA-induced dopamine release could be produced by the activation of a wide range of striatal VSCC located at the synaptic terminals and axons of striatal dopaminergic neurons, especially N-type VSCC.

Presynaptic adenosine receptor heteromers as key modulators of glutamatergic and dopaminergic neurotransmission in the striatum.

Adenosine plays a very significant role in modulating striatal glutamatergic and dopaminergic neurotransmission. In the present essay we first review the extensive evidence that indicates this modulation is mediated by adenosine A1 and A2A receptors (A1Rs and A2ARs) differentially expressed by the components of the striatal microcircuit that include cortico-striatal glutamatergic and mesencephalic dopaminergic terminals, and the cholinergic interneuron. This microcircuit mediates the ability of striatal glutamate release to locally promote dopamine release through the intermediate activation of cholinergic interneurons. A1Rs and A2ARs are colocalized in the cortico-striatal glutamatergic terminals, where they form A1R-A2AR and A2AR-cannabinoid CB1 receptor (CB1R) heteromers. We then evaluate recent findings on the unique properties of A1R-A2AR and A2AR-CB1R heteromers, which depend on their different quaternary tetrameric structure. These properties involve different allosteric mechanisms in the two receptor heteromers that provide fine-tune modulation of adenosine and endocannabinoid-mediated striatal glutamate release. Finally, we evaluate the evidence supporting the use of different heteromers containing striatal adenosine receptors as targets for drug development for neuropsychiatric disorders, such as Parkinson's disease and restless legs syndrome, based on the ability or inability of the A2AR to demonstrate constitutive activity in the different heteromers, and the ability of some A2AR ligands to act preferentially as neutral antagonists or inverse agonists, or to have preferential affinity for a specific A2AR heteromer.

The Anti-Aggregative Peptide KLVFF Mimics Aß1-40 in the Modulation of Nicotinic Receptors: Implications for Peptide-Based Therapy.

In recent years, the inhibition of beta-amyloid (Aß) aggregation has emerged as a potential strategy for Alzheimer's disease. KLVFF, a small peptide corresponding to the aminoacidic sequence 16-20 of Aß, reduces Aß fibrillation dose dependently. Therefore, the toxic and functional characterization of its brain activity is fundamental for clarifying its potential therapeutic role. Accordingly, we studied the modulatory role of KLVFF on the cholinergic receptors regulating dopamine and noradrenaline release in rat synaptosomes. Nicotinic receptors on dopaminergic nerve terminals in the nucleus acccumbens are inhibited by KLVFF, which closely resembles full-length Aß1-40. Moreover, KLVFF entrapped in synaptosomes does not modify the nicotinic receptor's function, suggesting that external binding to the receptor is required for its activity. The cholinergic agent desformylflustrabromine counteracts the KLVFF effect. Remarkably, muscarinic receptors on dopaminergic terminals and nicotinic receptors regulating noradrenaline release in the hippocampus are completely insensitive to KLVFF. Based on our findings, KLVFF mimics Aß1-40 as a negative modulator of specific nicotinic receptor subtypes affecting dopamine transmission in the rat brain. Therefore, new pharmacological strategies using the anti-aggregative properties of KLVFF need to be evaluated for potential interference with nicotinic receptor-mediated transmission.

Synaptotagmin 1-mediated cell membrane penetration and dopamine release enhancement by latroeggtoxin-VI.

Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin mined from the egg transcriptome of spider L. tredecimguttatus, was previously found to promote the release of dopamine from PC12 cells. However, the relevant molecular mechanism has not been fully clear. Here LETX-VI was demonstrated to rapidly penetrate the plasma membrane of PC12 cells via the vesicle exocytosis/endocytosis cycle, during which vesicular transmembrane protein synaptotagmin 1 (Syt1) functions as a receptor, with its vesicle luminal domain interacting with the C-terminal region of LETX-VI. The C-terminal sequence of LETX-VI is the functional region for both entering cells and promoting dopamine release. After gaining entry into the PC12 cells, LETX-VI down-regulated the phosphorylation levels of Syt1 at T201 and T195, thereby facilitating vesicle fusion with plasma membrane and thus promoting dopamine release. The relevant mechanism analysis indicated that LETX-VI has a protein phosphatase 2A (PP2A) activator activity. The present work has not only probed into the Syt1-mediated action mechanism of LETX-VI, but also revealed the structure-function relationship of the toxin, thus suggesting its potential applications in the drug transmembrane delivery and treatment of the diseases related to dopamine release and PP2A activity deficiency.

Fast-Scan Cyclic Voltammetry (FSCV) Reveals Behaviorally Evoked Dopamine Release by Sugar Feeding in the Adult Drosophila Mushroom Body.

Drosophila melanogaster, the fruit fly, is an excellent model organism for studying dopaminergic mechanisms and simple behaviors, but methods to measure dopamine during behavior are needed. Here, we developed fast-scan cyclic voltammetry (FSCV) to track in vivo dopamine during sugar feeding. First, we employed acetylcholine stimulation to evaluate the feasibility of in vivo measurements in an awake fly. Next, we tested sugar feeding by placing sucrose solution near the fly proboscis. In the mushroom body medial tip, 1 pmol acetylcholine and sugar feeding released 0.49±0.04 µM and 0.31±0.06 µM dopamine, respectively but sugar-evoked release lasted longer than with acetylcholine. Administering the dopamine transporter inhibitor nisoxetine or D2 receptor antagonist flupentixol significantly increased sugar-evoked dopamine. This study develops FSCV to measure behaviorally evoked release in fly, enabling Drosophila studies of neurochemical control of reward, learning, and memory behaviors.