RESUMO
Enzymes conjugated to nanomaterials are used in the design of various biotechnologies. In the development of biosensors, surface modifications with the enzyme glucose oxidase (GOx) serve to aid the detection of blood glucose. In order to optimize sensor effectiveness, the enzyme tertiary structure needs to be preserved upon immobilization to retain the enzyme's catalytic activity. Because of the nature of GOx, it suffers from a tendency to denature when immobilized at a solid surface; hence, methods to optimize enzyme stability are of great importance. Here, we introduce the study of the interaction of GOx to the highly curved surface of 20 nm gold nanoparticles (AuNP) with an absorbed monolayer coating of enzyme as determined by flocculation assays and quantification of immobilized GOx at the nanoparticle surface. Enzyme crowding was determined by comparing the number of enzymes that bind to how many can physically fit. These measurements show how placing a monolayer of enzyme where the enzyme spreads thin at the AuNP surface still provides stable catalytic performance of up to 14 days compared to enzymes free in solution. Moreover, by the increasing enzyme density via increasing the amount of GOx present in solution during the GOx/AuNP conjugation step creates a molecularly crowded environment at the highly curved nanoparticle surface. This limits the size of the enzyme footprint for attachment and shows that the activity per enzyme can be enhanced up to 300%. This is of great importance for implementing stable and sensitive sensor technologies that are constructed by enzyme-based nanoparticle scaffolds. Here, we show by using the conditions that maintain GOx structure and function when limiting the enzyme coating to an ultrathin layer, the design and construction of an ultrafast responding diagnostic sensor technology for glucose can be achieved, which is crucial for monitoring rapid fluctuations of, for instance, glucose in the brain.
Assuntos
Glucose Oxidase/química , Aspergillus niger/enzimologia , Estabilidade Enzimática , Glucose Oxidase/metabolismo , Ouro/química , Nanopartículas Metálicas/químicaRESUMO
Analytical tools for quantitative measurements of glutamate, the principal excitatory neurotransmitter in the brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for the counting of glutamate molecules stored inside single synaptic vesicles. In this method, an ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential-dependent manner at a constant negative potential. The continuous amperometric signals are sampled at high speed (10 kHz) to record sub-millisecond spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various glutamate concentrations. Our measurements show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis, and drug treatments for neuronal disorders where glutamate is involved.
Assuntos
Aminoácido Oxirredutases/química , Técnicas Eletroquímicas/métodos , Ácido Glutâmico/análise , Neurotransmissores/análise , Vesículas Sinápticas/química , Animais , Química Encefálica , Carbono/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ácido Glutâmico/química , Ouro/química , Masculino , Nanopartículas Metálicas/química , Camundongos Endogâmicos C57BL , Neurotransmissores/química , Ratos Sprague-Dawley , Lipossomas Unilamelares/químicaRESUMO
Psychostimulant use disorders remain an unabated public health concern worldwide, but no FDA approved medications are currently available for treatment. Modafinil (MOD), like cocaine, is a dopamine reuptake inhibitor and one of the few drugs evaluated in clinical trials that has shown promise for the treatment of cocaine or methamphetamine use disorders in some patient subpopulations. Recent structure-activity relationship and preclinical studies on a series of MOD analogs have provided insight into modifications of its chemical structure that may lead to advancements in clinical efficacy. Here, we have tested the effects of the clinically available (R)-enantiomer of MOD on extracellular dopamine levels in the nucleus accumbens shell, a mesolimbic dopaminergic projection field that plays significant roles in various aspects of psychostimulant use disorders, measured in vivo by fast-scan cyclic voltammetry and by microdialysis in Sprague-Dawley rats. We have compared these results with those obtained under identical experimental conditions with two novel and enantiopure bis(F) analogs of MOD, JBG1-048 and JBG1-049. The results show that (R)-modafinil (R-MOD), JBG1-048, and JBG1-049, when administered intravenously with cumulative drug-doses, will block the dopamine transporter and reduce the clearance rate of dopamine, increasing its extracellular levels. Differences among the compounds in their maximum stimulation of dopamine levels, and in their time course of effects were also observed. These data highlight the mechanistic underpinnings of R-MOD and its bis(F) analogs as pharmacological tools to guide the discovery of novel medications to treat psychostimulant use disorders.
Assuntos
Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Dopamina/farmacologia , Modafinila/farmacologia , Animais , Compostos Benzidrílicos/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Condicionamento Operante/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Masculino , Microdiálise/métodos , Núcleo Accumbens/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.
RESUMO
Hybrid structures constructed from biomolecules and nanomaterials have been used in catalysis and bioanalytical applications. In the design of many chemically selective biosensors, enzymes conjugated to nanoparticles or carbon nanotubes have been used in functionalization of the sensor surface for enhancement of the biosensor functionality and sensitivity. The conditions for the enzyme:nanomaterial conjugation should be optimized to retain maximal enzyme activity, and biosensor effectiveness. This is important as the tertiary structure of the enzyme is often altered when immobilized and can significantly alter the enzyme catalytic activity. Here we show that characterization of a two-enzyme:gold nanoparticle (AuNP) conjugate stoichiometry and activity can be used to gauge the effectiveness of acetylcholine detection by acetylcholine esterase (AChE) and choline oxidase (ChO). This was done by using an analytical approach to quantify the number of enzymes bound per AuNP and monitor the retained enzyme activity after the enzyme:AuNP synthesis. We found that the amount of immobilized enzymes differs from what would be expected from bulk solution chemistry. This analysis was further used to determine the optimal ratio of AChE:ChO added at synthesis to achieve optimum sequential enzyme activity for the enzyme:AuNP conjugates, and reaction efficiencies of greater than 70%. We here show that the knowledge of the conjugate stoichiometry and retained enzyme activity can lead to more efficient detection of acetylcholine by controlling the AChE:ChO ratio bound to the gold nanoparticle material. This approach of optimizing enzyme gold nanoparticle conjugates should be of great importance in the architecture of enzyme nanoparticle based biosensors to retain optimal sensor sensitivity.
Assuntos
Acetilcolinesterase/metabolismo , Oxirredutases do Álcool/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Acetilcolinesterase/química , Oxirredutases do Álcool/química , Técnicas Biossensoriais , EstereoisomerismoRESUMO
During exocytosis, small quantities of neurotransmitters are released by the cell. These neurotransmitters can be detected quantitatively using electrochemical methods, principally with disk carbon fiber microelectrode amperometry. An exocytotic event then results in the recording of a current peak whose characteristic features are directly related to the mechanisms of exocytosis. We have compared two exocytotic peak populations obtained from PC12 cells with a disk carbon fiber microelectrode and with a pyrolyzed carbon ring microelectrode array, with a 500 nm ring thickness. The specific shape of the ring electrode allows for precise analysis of diffusion processes at the vicinity of the cell membrane. Peaks obtained with a ring microelectrode array show a distorted average shape, owing to increased diffusion pathways. This result has been used to evaluate the diffusion coefficient of dopamine at the surface of a cell, which is up to an order of magnitude smaller than that measured in free buffer. The lower rate of diffusion is discussed as resulting from interactions with the glycocalyx.
Assuntos
Carbono/química , Membrana Celular/química , Dopamina/análise , Técnicas Eletroquímicas/métodos , Animais , Fibra de Carbono , Membrana Celular/metabolismo , Difusão , Dopamina/metabolismo , Técnicas Eletroquímicas/instrumentação , Microeletrodos , Células PC12 , Distribuição Aleatória , Ratos , Propriedades de SuperfícieRESUMO
Typical and atypical dopamine uptake inhibitors (DUIs) prefer distinct conformations of the dopamine transporter (DAT) to form ligand-transporter complexes, resulting in markedly different effects on behavior, neurochemistry, and potential for addiction. Here we show that cocaine and cocaine-like typical psychostimulants elicit changes in DA dynamics distinct from those elicited by atypical DUIs, as measured via voltammetry procedures. While both classes of DUIs reduced DA clearance rate, an effect significantly related to their DAT affinity, only typical DUIs elicited a significant stimulation of evoked DA release, an effect unrelated to their DAT affinity, which suggests a mechanism of action other than or in addition to DAT blockade. When given in combination, typical DUIs enhance the stimulatory effects of cocaine on evoked DA release while atypical DUIs blunt them. Pretreatments with an inhibitor of CaMKIIα, a kinase that interacts with DAT and that regulates synapsin phosphorylation and mobilization of reserve pools of DA vesicles, blunted the effects of cocaine on evoked DA release. Our results suggest a role for CaMKIIα in modulating the effects of cocaine on evoked DA release without affecting cocaine inhibition of DA reuptake. This effect is related to a specific DAT conformation stabilized by cocaine. Moreover, atypical DUIs, which prefer a distinct DAT conformation, blunt cocaine's neurochemical and behavioral effects, indicating a unique mechanism underlying their potential as medications for treating psychostimulant use disorder.
Assuntos
Estimulantes do Sistema Nervoso Central , Cocaína , Cocaína/farmacologia , Dopamina , Proteínas da Membrana Plasmática de Transporte de Dopamina , Inibidores da Captação de Dopamina/farmacologia , Proteínas Quinases Dependentes de Cálcio-CalmodulinaRESUMO
Fabrication of carbon microelectrode arrays, with up to 15 electrodes in total tips as small as 10-50 µm, is presented. The support structures of microelectrodes were obtained by pulling multiple quartz capillaries together to form hollow capillary arrays before carbon deposition. Carbon ring microelectrodes were deposited by pyrolysis of acetylene in the lumen of these quartz capillary arrays. Each carbon deposited array tip was filled with epoxy, followed by beveling of the tip of the array to form a deposited carbon-ring microelectrode array (CRMA). Both the number of the microelectrodes in the array and the tip size are independently tunable. These CRMAs have been characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and electrogenerated chemiluminescence. Additionally, the electrochemical properties were investigated with steady-state voltammetry. In order to demonstrate the utility of these fabricated microelectrodes in neurochemistry, CRMAs containing eight microring electrodes were used for electrochemical monitoring of exocytotic events from single PC12 cells. Subcellular temporal heterogeneities in exocytosis (i.e. cold spots vs hot spots) were successfully detected with the CRMAs.
Assuntos
Carbono/química , Técnicas Eletroquímicas/instrumentação , Exocitose , Análise de Célula Única/instrumentação , Análise Serial de Tecidos/instrumentação , Animais , Microeletrodos , Microscopia Eletrônica de Varredura , Microtecnologia/métodos , Células PC12 , Ratos , Espectrometria por Raios XRESUMO
A great deal of research has been focused on unraveling the processes governing the exocytotic pathway and the extent of release during the process. Arguments abound for and against both the occurrence and significance of full release during exocytosis and partial release including kiss-and-run events. Several optical methods to directly observe the exocytosis process have been developed and here we focus on fluorescence methods and probes for this work. Although fluorescence imaging has been used for cell experiments for decades, in the last two decades a plethora of new approaches have arrived on the scene. These include application of new microscopy techniques, like total internal reflectance and stimulated emission depletion that are offering new ways to circumvent the limits of far field microscopy with a diffraction limit of 200 nm, and allow tracking of single synaptic vesicles. For selective imaging of synaptic vesicles the introduction of methods to stain the vesicular compartment has involved developing probes of the vesicular membrane and intravesicular solution, nanoparticle quantum dots that can be observed during exocytosis but not via the fusion pore, and fluorescent false neurotransmitters.
Assuntos
Exocitose , Corantes Fluorescentes/química , Microscopia de Fluorescência , Nanopartículas , Pontos QuânticosRESUMO
To understand the correlation between animal behaviors and the underlying neuronal circuits, it is important to monitor and record neurotransmission in the brain of freely moving animals. With the development of fiber photometry, based on genetically encoded biosensors, and novel electrochemical biosensors, it is possible to measure some key neuronal transmission events specific to cell types or neurotransmitters of interest with high temporospatial resolution. This review discusses the recent advances and achievements of these two techniques in the study of neurotransmission in animal models and how they can be used to complement other techniques in the neuroscientist's toolbox.
Assuntos
Comportamento Animal/fisiologia , Mapeamento Encefálico/métodos , Encéfalo/metabolismo , Tecnologia de Fibra Óptica/métodos , Neurônios/metabolismo , Fibras Ópticas , Fotometria/métodos , Animais , Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Imageamento por Ressonância Magnética/métodos , Transmissão SinápticaRESUMO
Despite the high prevalence of obesity, little is known about its potential impact on the pharmacokinetics of psychotropic drugs. In the course of investigating the role of the microRNA system on neuronal signaling, we found that mice lacking the translin/trax microRNA-degrading enzyme display an exaggerated locomotor response to amphetamine. As these mice display robust adiposity in the context of normal body weight, we checked whether this phenotype might reflect elevated brain levels of amphetamine. To assess this hypothesis, we compared plasma and brain amphetamine levels of wild type and Tsn KO mice. Furthermore, we checked the effect of diet-induced increases in adiposity on plasma and brain amphetamine levels in wild type mice. Brain amphetamine levels were higher in Tsn KO mice than in wild type littermates and correlated with adiposity. Analysis of the effect of diet-induced increases in adiposity in wild type mice on brain amphetamine levels also demonstrated that brain amphetamine levels correlate with adiposity. Increased adiposity displayed by Tsn KO mice or by wild type mice fed a high-fat diet correlates with elevated brain amphetamine levels. As amphetamine and its analogues are widely used to treat attention deficit disorder, which is associated with obesity, further studies are warranted to assess the impact of adiposity on amphetamine levels in these patients.
Assuntos
Adiposidade , Anfetamina , Tecido Adiposo , Anfetamina/farmacologia , Animais , Encéfalo , Dieta Hiperlipídica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , ObesidadeRESUMO
Cocaine binds to the dopamine (DA) transporter (DAT) to regulate cocaine reward and seeking behavior. Zinc (Zn2+) also binds to the DAT, but the in vivo relevance of this interaction is unknown. We found that Zn2+ concentrations in postmortem brain (caudate) tissue from humans who died of cocaine overdose were significantly lower than in control subjects. Moreover, the level of striatal Zn2+ content in these subjects negatively correlated with plasma levels of benzoylecgonine, a cocaine metabolite indicative of recent use. In mice, repeated cocaine exposure increased synaptic Zn2+ concentrations in the caudate putamen (CPu) and nucleus accumbens (NAc). Cocaine-induced increases in Zn2+ were dependent on the Zn2+ transporter 3 (ZnT3), a neuronal Zn2+ transporter localized to synaptic vesicle membranes, as ZnT3 knockout (KO) mice were insensitive to cocaine-induced increases in striatal Zn2+. ZnT3 KO mice showed significantly lower electrically evoked DA release and greater DA clearance when exposed to cocaine compared to controls. ZnT3 KO mice also displayed significant reductions in cocaine locomotor sensitization, conditioned place preference (CPP), self-administration, and reinstatement compared to control mice and were insensitive to cocaine-induced increases in striatal DAT binding. Finally, dietary Zn2+ deficiency in mice resulted in decreased striatal Zn2+ content, cocaine locomotor sensitization, CPP, and striatal DAT binding. These results indicate that cocaine increases synaptic Zn2+ release and turnover/metabolism in the striatum, and that synaptically released Zn2+ potentiates the effects of cocaine on striatal DA neurotransmission and behavior and is required for cocaine-primed reinstatement. In sum, these findings reveal new insights into cocaine's pharmacological mechanism of action and suggest that Zn2+ may serve as an environmentally derived regulator of DA neurotransmission, cocaine pharmacodynamics, and vulnerability to cocaine use disorders.
Assuntos
Cocaína , Dopamina , Animais , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Camundongos , Núcleo Accumbens/metabolismo , Transmissão Sináptica , ZincoRESUMO
We report the synthesis and characterization of bioconjugates in which the enzymes malate dehydrogenase (MDH) and/or citrate synthase (CS) were adsorbed to 30 nm diameter Au nanoparticles. Enzyme:Au stoichiometry and kinetic parameters (specific activity, k(cat), K(M), and activity per particle) were determined for MDH:Au, CS:Au, and three types of dual-activity MDH/CS:Au bioconjugates. For single-activity bioconjugates (MDH:Au and CS:Au), the number of enzyme molecules adsorbed per particle was dependent upon the enzyme concentration in solution, with multilayers forming at high enzyme:Au solution ratios. The specific activity of adsorbed enzyme increased with increasing number adsorbed per particle for CS:Au, but was less sensitive to stoichiometry for MDH:Au. Dual activity bioconjugates were prepared in three ways: (1) by adsorption of MDH followed by CS, (2) by adsorption of CS followed by MDH, and (3) by coadsorption of both enzymes from the same solution. The resulting bioconjugates differed substantially in the number of enzyme molecules adsorbed per particle, the specific activity of the adsorbed enzymes, and also the enzymatic activity per particle. Bioconjugates formed by adding CS to the Au nanoparticles before MDH was added exhibited higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also had 3-fold higher per-particle sequential activity for conversion of malate to citrate, despite substantially fewer copies of both enzymes present.
Assuntos
Citrato (si)-Sintase/química , Citrato (si)-Sintase/metabolismo , Ouro/química , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Nanopartículas Metálicas/química , Adsorção , Animais , Domínio Catalítico , Corantes Fluorescentes/química , Cinética , Tamanho da PartículaRESUMO
Neuronal transmission relies on electrical signals and the transfer of chemical signals from one neuron to another. Chemical messages are transmitted from presynaptic neurons to neighboring neurons through the triggered fusion of neurotransmitter-filled vesicles with the cell plasma membrane. This process, known as exocytosis, involves the rapid release of neurotransmitter solutions that are detected with high affinity by the postsynaptic neuron. The type and number of neurotransmitters released and the frequency of vesicular events govern brain functions such as cognition, decision making, learning, and memory. Therefore, to understand neurotransmitters and neuronal function, analytical tools capable of quantitative and chemically selective detection of neurotransmitters with high spatiotemporal resolution are needed. Electrochemistry offers powerful techniques that are sufficiently rapid to allow for the detection of exocytosis activity and provides quantitative measurements of vesicle neurotransmitter content and neurotransmitter release from individual vesicle events. In this review, we provide an overview of the most commonly used electrochemical methods for monitoring single-vesicle events, including recent developments and what is needed for future research.
RESUMO
Modafinil and methylphenidate are medications that inhibit the neuronal reuptake of dopamine, a mechanism shared with cocaine. Their use as "smart drugs" by healthy subjects poses health concerns and requires investigation. We show that methylphenidate, but not modafinil, maintained intravenous self-administration in Sprague-Dawley rats similar to cocaine. Both modafinil and methylphenidate pretreatments potentiated cocaine self-administration. Cocaine, at self-administered doses, stimulated mesolimbic dopamine levels. This effect was potentiated by methylphenidate, but not by modafinil pretreatments, indicating dopamine-dependent actions for methylphenidate, but not modafinil. Modafinil is known to facilitate electrotonic neuronal coupling by actions on gap junctions. Carbenoxolone, a gap junction inhibitor, antagonized modafinil, but not methylphenidate potentiation of cocaine self-administration. Our results indicate that modafinil shares mechanisms with cocaine and methylphenidate but has a unique pharmacological profile that includes facilitation of electrotonic coupling and lower abuse liability, which may be exploited in future therapeutic drug design for cocaine use disorder.
Assuntos
Estimulantes do Sistema Nervoso Central , Cocaína , Metilfenidato , Animais , Compostos Benzidrílicos/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Dopamina , Relação Dose-Resposta a Droga , Junções Comunicantes , Metilfenidato/farmacologia , Modafinila , Ratos , Ratos Sprague-DawleyRESUMO
Atypical dopamine uptake inhibitors (DUIs) bind to the dopamine transporter and inhibit the reuptake of dopamine but have lower abuse potential than psychostimulants. Several atypical DUIs can block abuse-related effects of cocaine and methamphetamine, thus making them potential medication candidates for psychostimulant use disorders. The aim of the current study is to establish an in-vivo assay using EEG for the rapid identification of atypical DUIs with potential for medication development. The typical DUIs cocaine and methylphenidate dose-dependently decreased the power of the alpha, beta, and gamma bands. The atypical DUI modafinil and its F-analog, JBG1-049, decreased the power of beta, but in contrast to cocaine, none of the other frequency bands, while JHW007 did not significantly alter the EEG spectrum. The mu-opioid receptor agonists heroin and morphine dose-dependently decreased the power of gamma and increased power of the other bands. The effect of morphine on EEG power bands was antagonized by naltrexone. The NMDA receptor antagonist ketamine increased the power of all frequency bands. Therefore, typical and atypical DUIs and drugs of other classes differentially affected EEG spectra, showing distinctive features in the magnitude and direction of their effects on EEG. Comparative analysis of the effects of test drugs on EEG indicates a potential atypical profile of JBG1-049 with similar potency and effectiveness to its parent compound modafinil. These data suggest that EEG can be used to rapidly screen compounds for potential activity at specific pharmacological targets and provide valuable information for guiding the early stages of drug development. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Assuntos
Fármacos do Sistema Nervoso Central/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Eletroencefalografia/efeitos dos fármacos , Animais , Cocaína/farmacologia , Relação Dose-Resposta a Droga , Descoberta de Drogas , Heroína/farmacologia , Ketamina/farmacologia , Masculino , Metilfenidato/farmacologia , Modafinila/farmacologia , Morfina/farmacologia , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Entorpecentes/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/agonistasRESUMO
Neuronal communication relies on vesicular neurotransmitter release from signaling neurons and detection of these molecules by neighboring neurons. Glutamate, the main excitatory neurotransmitter in the mammalian brain, is involved in nearly all brain functions. However, glutamate has suffered from detection schemes that lack temporal and spatial resolution allowed by electrochemistry. Here we show an amperometric, novel, ultrafast enzyme-based nanoparticle modified sensor, measuring random bursts of hundreds to thousands of rapid spontaneous glutamate exocytotic release events at approximately 30 Hz frequency in the nucleus accumbens of rodent brain slices. Characterizing these single submillisecond exocytosis events revealed a great diversity in spike shape characteristics and size of quantal release, suggesting variability in fusion pore dynamics controlling the glutamate release by cells in this brain region. Hence, this novel biosensor allows recording of rapid single glutamate exocytosis events in the brain tissue and offers insight on regulatory aspects of exocytotic glutamate release, which is critical to understanding of brain glutamate function and dysfunction.
Assuntos
Exocitose/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Neurônios/efeitos dos fármacos , Neurotransmissores/metabolismo , Animais , Técnicas Biossensoriais/métodos , Encéfalo/efeitos dos fármacos , Eletroquímica/métodos , Exocitose/fisiologia , CamundongosRESUMO
Recent discoveries have improved our understanding of the physiological and pathological roles of the dopamine transporter (DAT); however, only a few drugs are clinically available for DAT-implicated disorders. Among those drugs, modafinil (MOD) and its ( R)-enantiomer (R-MOD) have been used off-label as therapies for psychostimulant use disorders, but they have shown limited effectiveness in clinical trials. Recent preclinical studies on MOD and R-MOD have led to chemically modified structures aimed toward improving their neurobiological properties that might lead to more effective therapeutics for stimulant use disorders. This study examines three MOD analogues (JJC8-016, JJC8-088, and JJC8-091) with improved DAT affinities compared to their parent compound. These compounds were investigated for their effects on the neurochemistry (brain microdialysis and FSCV) and behavior (ambulatory activity) of male Swiss-Webster mice. Our data indicate that these compounds have dissimilar effects on tonic and phasic dopamine in the nucleus accumbens shell and variability in producing ambulatory activity. These results suggest that small changes in the chemical structure of a DAT inhibitor can cause compounds such as JJC8-088 to produce effects similar to abused psychostimulants like cocaine. In contrast, other compounds like JJC8-091 do not share cocaine-like effects and have a more atypical DAT-inhibitor profile, which may prove to be an advancement in the treatment of psychostimulant use disorders.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Microdiálise/métodos , Modafinila/química , Modafinila/farmacologia , Núcleo Accumbens/metabolismo , Animais , Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Masculino , Camundongos , Núcleo Accumbens/efeitos dos fármacos , EstereoisomerismoRESUMO
Medication-assisted treatments are unavailable to patients with cocaine use disorders. Efforts to develop potential pharmacotherapies have led to the identification of a promising lead molecule, JJC8-091, that demonstrates a novel binding mode at the dopamine transporter (DAT). Here, JJC8-091 and a structural analogue, JJC8-088, were extensively and comparatively assessed to elucidate neurochemical correlates to their divergent behavioral profiles. Despite sharing significant structural similarity, JJC8-088 was more cocaine-like, increasing extracellular DA concentrations in the nucleus accumbens shell (NAS) efficaciously and more potently than JJC8-091. In contrast, JJC8-091 was not self-administered and was effective in blocking cocaine-induced reinstatement to drug seeking. Electrophysiology experiments confirmed that JJC8-091 was more effective than JJC8-088 at inhibiting cocaine-mediated enhancement of DA neurotransmission. Further, when VTA DA neurons in DAT-cre mice were optically stimulated, JJC8-088 produced a significant leftward shift in the stimulation-response curve, similar to cocaine, while JJC8-091 shifted the curve downward, suggesting attenuation of DA-mediated brain reward. Computational models predicted that JJC8-088 binds in an outward facing conformation of DAT, similar to cocaine. Conversely, JJC8-091 steers DAT towards a more occluded conformation. Collectively, these data reveal the underlying molecular mechanism at DAT that may be leveraged to rationally optimize leads for the treatment of cocaine use disorders, with JJC8-091 representing a compelling candidate for development.
Assuntos
Cocaína/antagonistas & inibidores , Inibidores da Captação de Dopamina/farmacologia , Oxalatos/farmacologia , Piperazinas/farmacologia , Animais , Cocaína/farmacologia , Dopamina/metabolismo , Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Comportamento de Procura de Droga/efeitos dos fármacos , Masculino , Simulação de Acoplamento Molecular , Núcleo Accumbens/metabolismo , Ratos , Autoadministração , Transmissão Sináptica/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacosRESUMO
Acetylcholine is a highly abundant nonelectroactive neurotransmitter in the mammalian central nervous system. Neurochemical release occurs on the millisecond time scale, requiring a fast, sensitive sensor such as an enzymatic amperometric electrode. Typically, the enzyme used for enzymatic electrochemical sensors is applied in excess to maximize signal. Here, in addition to sensitivity, we have also sought to maximize temporal resolution, by designing a sensor that is sensitive enough to work at near monolayer enzyme coverage. Reducing the enzyme layer thickness increases sensor temporal resolution by decreasing the distance and reducing the diffusion time for the enzyme product to travel to the sensor surface for detection. In this instance, the sensor consists of electrodeposited gold nanoparticle modified carbon fiber microelectrodes (CFMEs). Enzymes often are sensitive to curvature upon surface adsorption; thus, it was important to deposit discrete nanoparticles to maintain enzyme activity while depositing as much gold as possible to maximize enzyme coverage. To further enhance sensitivity, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized onto the gold nanoparticles at the previously determined optimal ratio (1:10 AChE/ChO) for most efficient sequential enzymatic activity. This optimization approach has enabled the rapid detection to temporally resolve single vesicle acetylcholine release from an artificial cell. The sensor described is a significant advancement in that it allows for the recording of acetylcholine release on the order of the time scale for neurochemical release in secretory cells.