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.

The three-dimensional nature of macromolecules is often difficult for undergraduate students to grasp. This leads to difficulties in understanding key concepts in Biochemistry, such as protein function and conformational change. Virtual reality (VR) technologies, which can aid students in three-dimensional visualizations, have been shown to increase student motivation, but published reports do not universally agree about whether VR improves student comprehension. Here we present the implementation of a VR experience that was designed to complement existing biochemistry experiments and an analysis of both student engagement with and understanding of the material presented in VR. Results indicate that students enjoyed this interactive, immersive activity and suggest evidence of increased understanding. However, the effectiveness of the VR experience-and effective assessment of such an experience-may depend on a number of factors. [GRAPHICS] .

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.

Cocaine binds to the dopamine (DA) transporter (DAT) to regulate cocaine reward and seeking behavior. Zinc (Zn 2+ ) also binds to the DAT, but the in vivo relevance of this interaction is unknown. We found that Zn 2+ 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 Zn 2+ 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 Zn 2+ concentrations in the caudate putamen (CPu) and nucleus accumbens (NAc). Cocaine-induced increases in Zn 2+ were dependent on the Zn 2+ transporter 3 (ZnT3), a neuronal Zn 2+ transporter localized to synaptic vesicle membranes, as ZnT3 knockout (KO) mice were insensitive to cocaine-induced increases in striatal Zn 2+ . 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 Zn 2+ deficiency in mice resulted in decreased striatal Zn 2+ content, cocaine locomotor sensitization, CPP, and striatal DAT binding. These results indicate that cocaine increases synaptic Zn 2+ release and turnover/metabolism in the striatum, and that synaptically-released Zn 2+ 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 Zn 2+ may serve as an environmentally-derived regulator of DA neurotransmission, cocaine pharmacodynamics, and vulnerability to cocaine use disorders.

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.

Modafinil and methylphenidate are among the few clinically available medications that block the dopamine (DA) transporter (DAT), a mechanism shared by abused psychostimulants like cocaine. Modafinil is FDA approved for the treatment of narcolepsy and other sleep disorders, while methylphenidate is approved as a medication for neurological disorders (e.g., attention deficit hyperactivity disorder). Both drugs have been reported for their non‐medical use as “smart drugs”, raising concerns regarding their potential for abuse by populations that may also be exposed to illicit substances. In this preclinical study, we compared the potential for abuse of modafinil with that of methylphenidate and their interactions with the reinforcing effects of cocaine in rats trained to self‐administer cocaine (0.03–1 mg/kg i.v., Fixed‐Ratio 5) with 5 doses assessed within each session. Since changes in DA levels in the nucleus accumbens shell (NAS) have been related to reinforcing effects of drugs, we monitored extracellular DA concentrations using microdialysis procedures in rats. Methylphenidate (0.03–1.0 mg/kg) maintained intravenous self‐administration behavior at comparable levels to active doses of cocaine (0.03–1.0 mg/kg). Modafinil (0.1–10 mg/kg) failed to maintain self‐administration behavior at any dose tested. However, pre‐session treatments (i.p.) with both modafinil (10–32 mg/kg) or methylphenidate (1.0–10 mg/kg) potentiated cocaine self‐administration behavior. Administration of cocaine, at self‐administered doses, produced dose‐related stimulation of NAS DA levels. Methylphenidate (1.0–10 mg/kg, i.p.), but not modafinil, (10–32 mg/kg, i.p.), enhanced cocaine‐induced stimulation of DA levels. These microdialysis results suggest that the effects of methylphenidate on cocaine actions are DA‐dependent, while those of modafinil are not. Modafinil is known to facilitate electrotonic coupling between cells by actions on gap junctions. Thus, we assessed the effects of carbenoxolone pretreatments, a gap junction inhibitor, on the potentiation of cocaine self‐administration produced by modafinil or methylphenidate. Carbenoxolone (1 mg/kg i.p.) attenuated modafinil, but not methylphenidate, potentiation of cocaine self‐administration behavior, suggesting that the facilitation of electrotonic coupling plays a role in modafinil’s potentiation of cocaine’s effects. In conclusion, modafinil shares with cocaine and methylphenidate important actions at the DAT, but the present data suggest a unique pharmacological stimulant profile, lacking abuse potential and facilitating electrotonic coupling. These results, together with clinical studies, further suggest a potential therapeutic use of modafinil in patients with cocaine use disorder.Support or Funding InformationThis research was supported by the Intramural Research Program of the National Institute on Drug Abuse, NIH, DHHS

The translin/trax microRNA-degrading enzyme mediates activity-induced changes in translation that underlie several long-lasting forms of cellular plasticity. As translin and trax are expressed in dopaminergic and striatal neurons, we investigated whether deletion of Tsn blocks amphetamine sensitization, a long-lasting, translation-dependent form of behavioral plasticity. Although we had hypothesized that constitutive Tsn deletion would impair amphetamine sensitization, we found, instead, that it enhances the hyperlocomotion produced by the initial dose of amphetamine. Since these mice display elevated adiposity, which alters pharmacokinetics of many drugs, we measured brain levels of amphetamine in Tsn knockout mice and found that these are elevated. As conditional Tsn deletion in adulthood does not impact adiposity, we monitored the locomotor response to amphetamine following this manipulation. Acute and sensitized responses to amphetamine are not altered in these mice, indicating that the enhanced amphetamine response displayed by constitutive Tsn knockout mice is due to Tsn absence during development.

Enzymes conjugated to nanomaterials are used in the design of various biotechnologies. In 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. Due to the nature of GOx, it suffers from tendency to denature when immobilized at a solid surface, 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) that shows how placing a monolayer of enzyme where the enzyme spreads thin at the AuNP surface still provides stable catalytic performance up to14 days compared to enzymes free in solution. Moreover, by increasing enzyme density and creating a molecularly crowded environment at the highly curved nanoparticle surface, which limits the size of the enzyme footprint for attachment, 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 ultra-thin layer, the design and construction of ultrafast responding diagnostic sensor technology for glucose can be achieved, which is crucial for monitoring rapid fluctuations of for instance, glucose in the brain.

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.

The translin/trax microRNA-degrading enzyme mediates activity-induced changes in translation that underlie several long-lasting forms of cellular plasticity. As translin and trax are expressed in dopaminergic and striatal neurons, we proceeded to investigate whether deletion of Tsn blocks amphetamine sensitization, a long-lasting, translation-dependent form of behavioral plasticity, Although we expected constitutive Tsn deletion to impair amphetamine sensitization, we found, instead, that it enhances the hyperlocomotion produced by the initial dose of amphetamine. Since these mice display elevated adiposity, which alters pharmacokinetics of many drugs, we measured brain amphetamine levels in Tsn knockout mice and found that these are elevated. We also found that diet-induced increases in adiposity in WT mice correlate 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 needed to assess the impact of adiposity on amphetamine levels in these patients.

Analytical tools for direct quantitative measurements of glutamate, the principal excitatory neurotransmitter in brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for direct counting of the number of glutamate molecules stored inside single synaptic vesicles. 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 by applying a constant negative potential. High-speed (10 kHz) amperometry is used to record sub-millisecond current 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 concentrations of glutamate. Our measurements show that a single synaptic vesicle encapsulates about 8000 glutamate molecules, which is comparable to the measured exocytotic quantal glutamate release 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.

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.

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.

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.

Dopamine uptake inhibitors (DUIs), such as cocaine and methylphenidate, are characterized by their ability to bind to the dopamine transporter (DAT) and prevent dopamine (DA) from being transported from the extracellular to the intracellular side of a neuronal terminal. It was originally posited that all DUIs would have the same effect on behavior, neurochemistry, and abuse liability in animal models as well as humans. However, a subset of these drugs, atypical DUIs (e.g. JHW007), have shown dissimilar profiles. While these DUIs have the same target and similar binding affinities to typical DUIs, their effects on behavior and neurochemistry are markedly different and predict little to no abuse liability in animal models.The goal of the present study was to determine how the neurochemistry of this atypical subset of DUIs differs from typical DUIs in mice. A pharmacological approach was taken utilizing Fast Scan Cyclic Voltammetry (FSCV), for real time monitoring of DA release and reuptake from the terminal; Fiber Photometry (FP), for real time monitoring of Ca2+ fluctuations in the terminal; and Electrophysiological (Ephys) ligand displacement experiments, to characterize the interactions between DUIs and the DAT.FSCV data indicate that cocaine and other abused DUIs have effects on both DA reuptake (by binding to the DAT and blocking DA transport) and stimulated release (causing an increase in mobilization of DA vesicles). In contrast the atypical DUIs, such as JHW007, have similar effects on DA reuptake, but do not cause a significant increase in stimulated DA release. FP experiments show how this difference influences Ca2+ fluctuations. JHW007 has a greater dose‐dependent impact on the influx of Ca2+ following stimulation than cocaine. Further Ephys experiments indicate a correlation between DA release and the off‐rate for each DUI, while no significant correlation was found for DA release and DAT affinity. Our data suggest 1) the dopaminergic effects of cocaine and other typical DUIs are not solely related to their ability to block the DAT; 2) DAT affinity alone is not an indicator for abuse liability; 3) the effect of the DUIs on the quaternary structure of the DAT and its downstream interactions may play an important role in the effects of typical DUIs related to abuse. Our preclinical results suggest that atypical DUIs can mitigate the effects of cocaine, leading to possible treatment candidates for psychostimulant use disorders in humans.Support or Funding InformationThis research was funded in part by the Medication Development Program, National Institute on Drug Abuse, IRP, NIH/DHHSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

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.

Modafinil has been used off‐label to treat psychostimulant use disorders (PSUD). However, its effectiveness in clinical trials seems limited to a subpopulation of subjects without concurrent alcohol or poly‐drug use. Since the main pharmacological target of cocaine is the dopamine transporter (DAT), recent efforts to develop more globally effective pharmacotherapies for PSUD have focused on two new DAT inhibitors and structural analogs of modafinil, JJC8‐088 and JJC8‐091, as promising lead compounds.In this study, the behavioral effects of these compounds were tested in rat models of cocaine self‐administration and reinstatement to drug seeking. Neurochemical correlates to those behaviors were assessed in microdialysis and fast‐scan cyclic voltammetry (FSCV) procedures aimed to measure extracellular dopamine levels and potential changes in dopamine dynamics in the nucleus accumbens shell (NAS).Our results show that JJC8‐088 (3, 10, 30 mg/kg, i.p.) dose‐dependently decreased the number of cocaine infusions under a fixed ratio schedule of self‐administration, while JJC8‐091 (10, 30, 56 mg/kg) failed to significantly alter cocaine self‐administration. In contrast, JJC8‐088 failed, while JJC8‐091 effectively decreased the break‐point for cocaine self‐administration under a progressive ratio schedule of behavior. JJC8‐088 maintained self‐administration behavior in both drug‐naïve and cocaine trained rats, while JJC8‐091 did not. Moreover, only priming injections of JJC8‐088 dose‐dependently reinstated drug‐seeking behavior in rats following previous cocaine extinction. Importantly, in the same rats, JJC8‐091 pretreatment blocked cocaine‐induced reinstatement of drug seeking behavior.The neurochemical assessment of JJC8‐088 and JJC8‐091 effects showed that both drugs dose‐dependently increased extracellular DA concentrations in dialysates from the NAS. However, JJC8‐088 was more potent and more efficacious in increasing DA levels compared to JJC8‐091. Indeed, the maximal changes in DA levels elicited by JJC8‐088 resemble those obtained with cocaine, in contrast to JJC8‐091.The FSCV studies showed that both JJC8‐088 and JJC8‐091 dose dependently reduced the rate of DA clearance, with JJC8‐088 producing effects greater in magnitude at lower doses than JJC8‐091. Finally, like cocaine, cumulative doses of JJC8‐088 produced a dose‐dependent increase in DAMax, while JJC8‐091 was ineffective at all doses tested.Collectively, these data show that despite sharing significant structural similarity, JJC8‐088 was cocaine‐like, while JJC8‐091 was not. Indeed, in contrast with JJC8‐088, JJC8‐091 did not elicit significant reinforcing actions and reduced or blunted the reinforcing effects of cocaine. Taken together, our data support further development of JJC8‐091, as a potential pharmacotherapy for the treatment of PSUD.Support or Funding InformationSupport for this research was provided by the Medication Development Program, National Institute on Drug Abuse ‐ Intramural Research Program, NIH/DHHSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

0 0 1 149 852 Gothenburg University 7 1 1000 14.0 Normal 0 false false false EN-US JA X-NONE /∗ Style Definitions ∗/ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin;} 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 ultra-fast enzyme-based nanoparticle modified sensor, measuring bursts of hundreds to thousands of rapid spontaneous glutamate exocytotic release events at approximately 30 Hz frequency in rodent brain slices. Characterizing these single sub-millisecond 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 these neurons. 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 in dysfunction.