Omar S. Mabrouk

Mesolimbic dopamine signals the value of work

Dopamine cell firing can encode errors in reward prediction, providing a learning signal to guide future behavior. Yet dopamine is also a key modulator of motivation, invigorating current behavior. Existing theories propose that fast (phasic) dopamine fluctuations support learning, whereas much slower (tonic) dopamine changes are involved in motivation. We examined dopamine release in the nucleus accumbens across multiple time scales, using complementary microdialysis and voltammetric methods during adaptive decision-making. We found that minute-by-minute dopamine levels covaried with reward rate and motivational vigor. Second-by-second dopamine release encoded an estimate of temporally discounted future reward (a value function). Changing dopamine immediately altered willingness to work and reinforced preceding action choices by encoding temporal-difference reward prediction errors. Our results indicate that dopamine conveys a single, rapidly evolving decision variable, the available reward for investment of effort, which is employed for both learning and motivational functions.

In Vivo Neurochemical Monitoring Using Benzoyl Chloride Derivatization and Liquid Chromatography–Mass Spectrometry

In vivo neurochemical monitoring using microdialysis sampling is important in neuroscience because it allows correlation of neurotransmission with behavior, disease state, and drug concentrations in the intact brain. A significant limitation of current practice is that different assays are utilized for measuring each class of neurotransmitter. We present a high performance liquid chromatography (HPLC)-tandem mass spectrometry method that utilizes benzoyl chloride for determination of the most common low molecular weight neurotransmitters and metabolites. In this method, 17 analytes were separated in 8 min. The limit of detection was 0.03-0.2 nM for monoamine neurotransmitters, 0.05-11 nM for monoamine metabolites, 2-250 nM for amino acids, 0.5 nM for acetylcholine, 2 nM for histamine, and 25 nM for adenosine at sample volume of 5 μL. Relative standard deviation for repeated analysis at concentrations expected in vivo averaged 7% (n = 3). Commercially available (13)C benzoyl chloride was used to generate isotope-labeled internal standards for improved quantification. To demonstrate utility of the method for study of small brain regions, the GABA(A) receptor antagonist bicuculline (50 μM) was infused into a rat ventral tegmental area while recording neurotransmitter concentration locally and in nucleus accumbens, revealing complex GABAergic control over mesolimbic processes. To demonstrate high temporal resolution monitoring, samples were collected every 60 s while neostigmine, an acetylcholine esterase inhibitor, was infused into the medial prefrontal cortex. This experiment revealed selective positive control of acetylcholine over cortical glutamate.

Forebrain deletion of the dystonia protein torsinA causes dystonic-like movements and loss of striatal cholinergic neurons

Striatal dysfunction plays an important role in dystonia, but the striatal cell types that contribute to abnormal movements are poorly defined. We demonstrate that conditional deletion of the DYT1 dystonia protein torsinA in embryonic progenitors of forebrain cholinergic and GABAergic neurons causes dystonic-like twisting movements that emerge during juvenile CNS maturation. The onset of these movements coincides with selective degeneration of dorsal striatal large cholinergic interneurons (LCI), and surviving LCI exhibit morphological, electrophysiological, and connectivity abnormalities. Consistent with the importance of this LCI pathology, murine dystonic-like movements are reduced significantly with an antimuscarinic agent used clinically, and we identify cholinergic abnormalities in postmortem striatal tissue from DYT1 dystonia patients. These findings demonstrate that dorsal LCI have a unique requirement for torsinA function during striatal maturation, and link abnormalities of these cells to dystonic-like movements in an overtly symptomatic animal model. DOI: http://dx.doi.org/10.7554/eLife.08352.001

Rapid dopamine transmission within the nucleus accumbens: Dramatic difference between morphine and oxycodone delivery

While most drugs of abuse increase dopamine neurotransmission, rapid neurochemical measurements show that different drugs evoke distinct dopamine release patterns within the nucleus accumbens. Rapid changes in dopamine concentration following psychostimulant administration have been well studied; however, such changes have never been examined following opioid delivery. Here, we provide novel measures of rapid dopamine release following intravenous infusion of two opioids, morphine and oxycodone, in drug‐naïve rats using fast‐scan cyclic voltammetry and rapid (1 min) microdialysis coupled with high‐performance liquid chromatography ‐ tandem mass spectrometry (HPLC‐MS). In addition to measuring rapid dopamine transmission, microdialysis HPLC‐MS measures changes in GABA, glutamate, monoamines, monoamine metabolites and several other neurotransmitters. Although both opioids increased dopamine release in the nucleus accumbens, their patterns of drug‐evoked dopamine transmission differed dramatically. Oxycodone evoked a robust and stable increase in dopamine concentration and a robust increase in the frequency and amplitude of phasic dopamine release events. Conversely, morphine evoked a brief (~ 1 min) increase in dopamine that was coincident with a surge in GABA concentration and then both transmitters returned to baseline levels. Thus, by providing rapid measures of neurotransmission, this study reveals previously unknown differences in opioid‐induced neurotransmitter signaling. Investigating these differences may be essential for understanding how these two drugs of abuse could differentially usurp motivational circuitry and powerfully influence behavior.

Benzoyl chloride derivatization with liquid chromatography-mass spectrometry for targeted metabolomics of neurochemicals in biological samples

Widely targeted metabolomic assays are useful because they provide quantitative data on large groups of related compounds. We report a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method that utilizes benzoyl chloride labeling for 70 neurologically relevant compounds, including catecholamines, indoleamines, amino acids, polyamines, trace amines, antioxidants, energy compounds, and their metabolites. The method includes neurotransmitters and metabolites found in both vertebrates and insects. This method was applied to analyze microdialysate from rats, human cerebrospinal fluid, human serum, fly tissue homogenate, and fly hemolymph, demonstrating its broad versatility for multiple physiological contexts and model systems. Limits of detection for most assayed compounds were below 10nM, relative standard deviations were below 10%, and carryover was less than 5% for 70 compounds separated in 20min, with a total analysis time of 33min. This broadly applicable method provides robust monitoring of multiple analytes, utilizes small sample sizes, and can be applied to diverse matrices. The assay will be of value for evaluating normal physiological changes in metabolism in neurochemical systems. The results demonstrate the utility of benzoyl chloride labeling with HPLC-MS/MS for widely targeted metabolomics assays.

Microdialysis and mass spectrometric monitoring of dopamine and enkephalins in the globus pallidus reveal reciprocal interactions that regulate movement

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07293.x

Ventral tegmental area neurotensin signaling links the lateral hypothalamus to locomotor activity and striatal dopamine efflux in male mice

Projections from the lateral hypothalamic area (LHA) innervate components of the mesolimbic dopamine (MLDA) system, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), to modulate motivation appropriately for physiologic state. Neurotensin (NT)-containing LHA neurons respond to multiple homeostatic challenges and project to the VTA, suggesting that these neurons could link such signals to MLDA function. Indeed, we found that pharmacogenetic activation of LHA NT neurons promoted prolonged DA-dependent locomotor activity and NAc DA efflux, suggesting the importance of VTA neurotransmitter release by LHA NT neurons for the control of MLDA function. Using a microdialysis-mass spectrometry technique that we developed to detect endogenous NT in extracellular fluid in the mouse brain, we found that activation of LHA NT cells acutely increased the extracellular concentration of NT (a known activator of VTA DA cells) in the VTA. In contrast to the prolonged elevation of extracellular NAc DA, however, VTA NT concentrations rapidly returned to baseline. Intra-VTA infusion of NT receptor antagonist abrogated the ability of LHA NT cells to increase extracellular DA in the NAc, demonstrating that VTA NT promotes NAc DA release. Thus, transient LHA-derived NT release in the VTA couples LHA signaling to prolonged changes in DA efflux and MLDA function.

Simultaneous oxytocin and arg-vasopressin measurements in microdialysates using capillary liquid chromatography–mass spectrometry

Oxytocin (OXT) and arg-vasopressin (AVP) are nonapeptides with many important functions both peripherally and centrally. Intracerebral microdialysis has helped characterize their importance in regulating complex social and emotional processes. Radioiummunoassay is the most commonly used analytical method used for OXT and AVP measurements in microdialysates. These measurements have several well-known issues including single peptide per assay limit, possible cross-reactivity between structurally related peptides, and laborious sample preparation with radioactive materials. Here we demonstrate the use of capillary LC-MS(3) for measuring OXT and AVP simultaneously in dialysates at a 10 min sampling frequency. Microdialysate samples required no preparation and instrumentation was commercially available. Microdialysis probes made with polyacrylonitrile membranes were suitable for high level recovery of the peptides in vitro and in vivo. Responses were linear from 1 to 100 pM. Matrix effect was assessed by standard addition experiments and by comparing signal intensities of OXT and AVP standards made in aCSF or dialysate. It was determined that the online washing step used on this setup was adequate for removing contaminants which interfere with electrospray ionization efficiency. In vivo, both peptides were stimulated by high K(+) (75 mM) aCSF perfusion in the paraventricular nucleus (PVN). Also, a systemic injection of high Na(+) (2M) caused a rapid and transient increase in PVN OXT while AVP increased only after 1.5h. Our findings suggest that capillary LC-MS(3) is a straightforward method for monitoring OXT and AVP simultaneously from complex samples such as dialysates.

Human Transgene-Free Amniotic-Fluid-Derived Induced Pluripotent Stem Cells for Autologous Cell Therapy

The establishment of a reliable prenatal source of autologous, transgene-free progenitor cells has enormous potential in the development of regenerative-medicine-based therapies for infants born with devastating birth defects. Here, we show that a largely CD117-negative population of human amniotic fluid mesenchymal stromal cells (AF-MSCs) obtained from fetuses with or without prenatally diagnosed anomalies are readily abundant and have limited baseline differentiation potential when compared with bone-marrow-derived MSCs and other somatic cell types. Nonetheless, the AF-MSCs could be easily reprogrammed into induced pluripotent stem cells (iPSCs) using nonintegrating Sendai viral vectors encoding for OCT4, SOX2, KLF4, and cMYC. The iPSCs were virtually indistinguishable from human embryonic stem cells in multiple assays and could be used to generate a relatively homogeneous population of neural progenitors, expressing PAX6, SOX2, SOX3, Musashi-1, and PSA-NCAM, for potential use in neurologic diseases. Further, these neural progenitors showed engraftment potential in vivo and were capable of differentiating into mature neurons and astrocytes in vitro. This study demonstrates the usefulness of AF-MSCs as an excellent source for the generation of human transgene-free iPSCs ideally suited for autologous perinatal regenerative medicine applications.

Chemical gradients within brain extracellular space measured using low flow push-pull perfusion sampling in vivo

Although populations of neurons are known to vary on the micrometer scale, little is known about whether basal concentrations of neurotransmitters also vary on this scale. We used low-flow push-pull perfusion to test if such chemical gradients exist between several small brain nuclei. A miniaturized polyimide-encased push-pull probe was developed and used to measure basal neurotransmitter spatial gradients within brain of live animals with 0.004 mm(3) resolution. We simultaneously measured dopamine (DA), norepinephrine, serotonin (5-HT), glutamate, γ-aminobutyric acid (GABA), aspartate (Asp), glycine (Gly), acetylcholine (ACh), and several neurotransmitter metabolites. Significant differences in basal concentrations between midbrain regions as little as 200 μm apart were observed. For example, dopamine in the ventral tegmental area (VTA) was 4.8 ± 1.5 nM but in the red nucleus was 0.5 ± 0.2 nM. Regions of high glutamate concentration and variability were found within the VTA of some individuals, suggesting hot spots of glutamatergic activity. Measurements were also made within the nucleus accumbens core and shell. Differences were not observed in dopamine and 5-HT in the core and shell; but their metabolites homovanillic acid (460 ± 60 nM and 130 ± 60 nM respectively) and 5-hydroxyindoleacetic acid (720 ± 200 nM and 220 ± 50 nM respectively) did differ significantly, suggesting differences in dopamine and 5-HT activity in these brain regions. Maintenance of these gradients depends upon a variety of mechanisms. Such gradients likely underlie highly localized effects of drugs and control of behavior that have been found using other techniques.