Viewpoint pubs.acs.org/chemneuro
Differentiating Siblings: The Case of Dopamine and Norepinephrine Nako Nakatsuka†,‡ and Anne M. Andrews*,†,‡,§ †
California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California 90095, United States Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States § Department of Psychiatry and Biobehavioral Health, Semel Institute for Neuroscience and Human Behavior, and Hatos Center for Neuropharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, United States ‡
ABSTRACT: Monitoring dopamine and norepinephrine (or other structurally similar neurotransmitters) in the same brain region necessitates selective sensing. In this Viewpoint, we highlight electrochemical and optical strategies for advancing simultaneous real-time measurements of dopamine and norepinephrine transmission. The potential for DNA aptamers as recognition elements in the context of field-effect transistor sensing for selective and simultaneous neurotransmitter monitoring in vivo is also discussed. KEYWORDS: Biosensors, fast-scan cyclic voltammetry, CNiFERs, frontal cortex, psychiatric disorders
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A highly localized anatomy coupled with ease of electrochemical detection largely underlies the predominance of neurochemical investigation of dopamine signaling. Norepinephrine is also detected electrochemically. In contrast to dopaminergic projections, noradrenergic axons, originating from brain stem locus coeruleus and medullary (A1/A2) neurons, project diffusely to most brain regions, including those innervated by the dopamine system. There are number of key areas of overlap between the dopamine and norepinephrine systems including the hypothalamus, bed nucleus of the stria terminalis (BNST), and frontal cortex. These brain regions process stress and emotion-related information. Dopamine and norepinephrine neurocircuits are implicated in modulating reward- and emotion-related behavior and, by association, the etiology and treatment of mood and anxiety disorders. To investigate causal relationships in brain disorders such as depression, we need neurochemical techniques for realtime monitoring that have sufficient chemical specificity and spatiotemporal resolution to differentiate dopamine and norepinephrine signaling. At present, two in vivo techniques are most often used to determine dopamine and norepinephrine levels in the extracellular space. Microdialysis is coupled to an analytical method like high performance liquid chromatography or mass spectrometry. By contrast, carbon-fiber microelectrode voltammetry is a direct measurement technique. Both approaches have limitations that hinder progress toward understanding brain function. Microdialysis enables multiplexing; therefore, both dopamine and norepinephrine can be monitored in the same brain regions. However, while throughput and sampling times are improving (DOI: 10.1021/acschemneuro.6b00383 this issue), temporal
t is no coincidence that neurotransmitters and other small molecules involved in biological signaling are closely structurally related. Evolution over millennia has imparted biological systems with finely tuned efficiency in the form of intertwined synthesis motifs. Amino acids are used as building blocks of proteins but also as neurotransmitters or their precursors. Glutamate is a prime example. Furthermore, decarboxylation of glutamate yields γ-aminobutyric acid (GABA). Together, glutamate and GABA are responsible for a majority of interneuronal communication in the central nervous system. Less abundant but no less important are the catecholamine neurotransmitters dopamine, norepinephrine, and epinephrine, synthesized from the amino acid tyrosine in stepwise fashion (Figure 1). Dopamine neurons are located in the midbrain substantia nigra and ventral tegmental area. These neurons project densely to the dorsal and ventral striatum, respectively.
Figure 1. Dopamine is synthesized in two steps from the amino acid tyrosine in dopaminergic neurons. Norepinephrine is produced from dopamine in noradrenergic neurons. Epinephrine is synthesized from norepinephrine in adrenergic neurons. Tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine-β-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). © XXXX American Chemical Society
Special Issue: Monitoring Molecules in Neuroscience 2016 Received: February 6, 2017 Accepted: February 6, 2017
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DOI: 10.1021/acschemneuro.7b00056 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
ACS Chemical Neuroscience
Viewpoint
local neurotransmitter activity. Using dopamine and norepinephrine CNiFERs implanted 300 μm apart in layer 2/3 of frontal cortex, Slesinger, Kleinfeld, and co-workers differentiated dopamine and norepinephrine responses during learning about the salience of a conditioned stimulus in mice. Moreover, CNiFERs were used to detect acetylcholine release associated with motor/licking behavior. Equilibrium sensing methods are typically limited to detection within an order of magnitude in either direction of receptor dissociation constants. Limitations of CNiFERs can be overcome by using GPCRs with different endogenous neurotransmitter affinities.3 Imaging subcortical structures may be possible via optical fibers or endoscopes in freely moving animals (DOI: 10.1021/acschemneuro.6b00307, this issue). Possibly, CNiFERs can be used to detect any neurotransmitter that signals via GPCRs by re-engineering G-protein coupling for Gq intracellular signaling. We are taking a somewhat different approach to overcoming challenges associated with temporally and spatially resolved multiplexed neurotransmitter detection. Instead of using native GPCRs, we employ short DNA oligomers or aptamers, which are nucleic acid sequences that fold into unique three-dimensional structures to recognize many types of molecules, including small-molecule neurotransmitters.4 For in vivo neurotransmitter monitoring, aptamers are highly attractive synthetic molecular recognition elements because they rival or surpass antibodies and native receptors in several key ways. They offer improved stability compared to proteins due to chemically robust phosphodiester backbones. High affinity aptamers can be generated through high stringency in vitro selection methods.4 Most importantly, for addressing the challenges of neurotransmitter signal differentiation, aptamers can be negatively selected to reduce cross-reactivity toward structurally similar targets, even those that differ by a single functional group, e.g., caffeine vs theophylline. Sensing dopamine and norepinephrine (and other neurotransmitters) requires not only high affinity and highly selective aptamers but a transduction method that embodies the spatiotemporal attributes needed for in vivo monitoring (response times < 1 s, μm to nm feature sizes), while detecting analytes over low and large concentration ranges. To address this, we have coupled aptamers to field-effect transistors (FETs) to enable detection of dopamine.5 We have also tested newer, higher affinity aptamers that discriminate dopamine and norepinephrine with high selectivity. In FETs, semiconductor channels are positioned between metal source- and drain-electrodes. The FET channels are functionalized with aptamers. Upon binding to neurotransmitters, the conformations of negatively charged DNA aptamers are altered and thereby induce changes in channel-surface electric fields. Sensing via FETs is nonlinear; even small changes in surface electric field measurably gate channel potentials and, thus, source-drain currents. In this way, small changes in aptamer occupancy lead to detectable sensor responses. This mechanism leads to highly sensitive and fast detection of neurotransmitter levels over 6 orders of magnitude. Individual FETs can be fabricated on silicon microprobes6 or other flexible devices at high densities and small sizes. When individual FETs are functionalized with different high-affinity aptamers, these devices will be advantageous for acute and chronic multiplexed intracerebral recordings. The capacity to monitor multiple neurotransmitters simultaneously in the presence of structurally similar precursors, metabolites, and other neurotransmitters
To investigate causal relationships in brain disorders such as depression, we need neurochemical techniques for real-time monitoring that have sufficient chemical specificity and spatiotemporal resolution to differentiate dopamine and norepinephrine signaling. and spatial resolution are not well suited to reveal the properties of complex neural circuits. Moreover, the implantation of microdialysis probes disrupts neuronal activity near the probe track. Fast-scan cyclic voltammetry (FSCV) enables improvements in spatiotemporal resolution. Nonetheless, it is difficult to differentiate dopamine and norepinephrine. Due to their high degree of structural similarity (Figure 1), dopamine and norepinephrine have overlapping redox potentials and thus, cyclic voltammograms. Multiplexed measurements of dopaminergic and noradrenergic signaling are difficult to implement to investigate the interplay between these chemical transmitters. Wightman, Carelli, and co-workers partly circumvented limitations associated with FSCV by monitoring dopamine and norepinephrine release in two closely located subregions of the BNST (150 μm apart).1 These investigators targeted carbon-fiber microelectrodes accurately to measure dopamine and norepinephrine in behaving rats. Catecholamine responses differed in response to stimuli with opposing valence. Dorsolateral BNST dopamine levels increased with a sucrose (rewarding) stimulus yet decreased when rats received intraoral quinine (an aversive stimulus). Ventral BNST norepinephrine levels were modulated oppositely, increasing after the aversive stimulus. This approach, while elegant and powerful, has not overcome the challenge of detecting dopamine and norepinephrine in the same brain region, e.g., frontal cortex, and at the same time in the same animals (to take advantage of within-subjects comparisons). Other approaches to characterizing chemical neurotransmission via optical methods are being developed that have the potential to improve multiplexed detection of structurally similar neurotransmitters. For example, a catecholamine fluorescence sensor differentiates primary amines, i.e., dopamine and norepinephrine, from secondary amines such as epinephrine (Figure 1). This sensor is based on a coumarin aldehyde scaffold and enables selective recognition of catecholamines in live and fixed cells.2 Norepinephrine-containing secretory vesicles were preferentially stained over epinephrine-containing vesicles differentiating two populations of chromaffin cells. Nonetheless, this type of sensor cannot yet distinguish dopamine and norepinephrine. Moreover, affinities are millimolar and thus higher than what will be needed for in vivo deployment and extracellular monitoring. A different approach to optical detection involves cell-based neurotransmitter fluorescent engineered reporters (CNiFERs).3 Genetically engineered HEK293 cells are implanted into the brain to detect neurotransmitter concentrations dynamically via two-photon microscopy. In CNiFER cells, neurotransmitter-specific binding to G-protein-coupled receptors (GPCRs) causes a Gq-mediated increase in intracellular Ca2+, which is transformed into a fluorescent signal using a FRET-based Ca2+ sensor. These CNiFERs provide real-time optical readouts of B
DOI: 10.1021/acschemneuro.7b00056 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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in vivo will enable elucidation of transmitter interactions within the context of understanding native behavior and behavior associated with neuropsychiatric and neurodegenerative disorders.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Anne M. Andrews: 0000-0002-1961-4833 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Park, J., Wheeler, R. A., Fontillas, K., Keithley, R. B., Carelli, R. M., and Wightman, R. M. (2012) Catecholamines in the bed nucleus of the stria terminalis reciprocally respond to reward and aversion. Biol. Psychiatry 71, 327−334. (2) Hettie, K. S., Liu, X., Gillis, K. D., and Glass, T. E. (2013) Selective catecholamine recognition with NeuroSensor 521: A fluorescent sensor for the visualization of norepinephrine in fixed and live cells. ACS Chem. Neurosci. 4, 918−923. (3) Muller, A., Joseph, V., Slesinger, P. A., and Kleinfeld, D. (2014) Cell-based reporters reveal in vivo dynamics of dopamine and norepinephrine release in murine cortex. Nat. Methods 11, 1245−1252. (4) Yang, K. A., Pei, R., and Stojanovic, M. N. (2016) In vitro selection and amplification protocols for isolation of aptameric sensors for small molecules. Methods 106, 58−65. (5) Kim, J., Rim, Y. S., Chen, H., Cao, H. H., Nakatsuka, N., Hinton, H. L., Zhao, C., Andrews, A. M., Yang, Y., and Weiss, P. S. (2015) Fabrication of high-performance ultrathin In2O3 film field-effect transistors and biosensors using chemical lift-off lithography. ACS Nano 9, 4572−4582. (6) Wassum, K. M., Tolosa, V. M., Tseng, T. C., Balleine, B. W., Monbouquette, H. G., and Maidment, N. T. (2012) Transient extracellular glutamate events in the basolateral amygdala track rewardseeking actions. J. Neurosci. 32, 2734−2746.
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DOI: 10.1021/acschemneuro.7b00056 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
