Article pubs.acs.org/accounts
Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry Published as part of the Accounts of Chemical Research special issue “Nanoelectrochemistry”. Xianchan Li,†,§ Johan Dunevall,‡,§ and Andrew G. Ewing*,†,‡ †
Department of Chemistry and Molecular Biology, University of Gothenburg, SE-405 30 Gothenburg, Sweden Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden
‡
CONSPECTUS: Electrochemical cytometry adds a new dimension to our ability to study the chemistry and chemical storage of transmitter molecules stored in nanometer vesicles. The approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which the electroactive contents are quantitatively oxidized (or reduced). The measured current allows us to count the number of molecules in the vesicles using Faraday’s law and to correlate this to the amount of molecules released when single exocytosis events take place at communicating cells. The original format for this method involved a capillary electrophoresis separation step to singly address each vesicle, but we have more recently discovered that cellular vesicles tend to adsorb to carbon electrodes and spontaneously as well as stochastically rupture to give mostly single vesicle events. This approach, called impact electrochemical cytometry, even though the impact is perhaps not the important part of this process, has been studied and the vesicle rupture appears to be at the interface between the vesicle and the electrode and is probably driven by electroporation. The pore size and rate of content electrolysis are a function of the pore diameter and the presence of a protein core in the vesicles. In model liposomes with no protein, events appear extremely rapidly as the soft nanoparticles impact the electrode and the contents are oxidized. It appears that the proteins decorating the surface of the vesicle are important in maintaining a gap from the electrode and when this gap is closed electroporation takes place. Models of the event response times suggest the pores formed are small enough so we can carry out these measurements at nanotip electrodes and we have used this to quantify the vesicle content in living cells in a mode we call intracellular impact electrochemical cytometry. The development of electrochemical cytometry allows comparison between vesicle content and vesicular release and we have found that only part of the vesicle content is released in typical exocytotic cases measured by amperometry. This has led to the novel hypothesis that most exocytosis from dense core vesicles is via mechanism where vesicles fuse with the cell membrane, some content is released and then close again to be reloaded and reused. It leaves open the possibility that cells regulate release during individual events. This might be important in learning and memory and be a nonreceptor pharmaceutical target for brainrelated disorders. Indeed, the concept of the chemo-brain observed in cisplatin-treated cancer patients appears to be at least in part the result of changing the fraction of transmitter released and we have been able to show this by using the combined amperometric measurement of release and electrochemical cytometry at model cells.
1. INTRODUCTION
synaptic vesicles are most common in neuronal synaptic transmission, and these vesicles are smaller in size compared to large dense core vesicles (approximately 50 nm vs 150−300 nm in diameter, respectively). Large dense core vesicles occur in some neuronal cells, but are most commonly found in neuroendocrine cells (e.g., pancreatic β-cells and adrenal chromaffin cells). In adrenal chromaffin cells the large dense core vesicles contain a protein matrix (dense core), which is mainly composed of proteins in the chromogranin family (CgA and CgB). These polyanionic proteins retain the cationic catecholamines, such as epinephrine and norepinephrine, at relatively high concentrations.2,3 The dense core plays two
Intercellular communication in the nervous system is mainly carried out through exocytosis.1 During exocytosis, vesicles loaded with neurotransmitters, neurohormones, and/or neuropeptides migrate to the cell membrane and through a series of steps are docked, primed, and then fuse with the plasma membrane. During fusion, a pore is formed through which the vesicular cargo is released into the extracellular space (e.g., synaptic cleft or the bloodstream), presumably through diffusion. Once released, these messenger molecules can interact with receptors at target cells across a synaptic gap, or sometimes a long distance away, causing cascades of reactions through which the signal is propagated. Secretory vesicles can generally be divided into two groups: clear synaptic vesicles and large dense core vesicles. The clear © 2016 American Chemical Society
Received: June 30, 2016 Published: September 13, 2016 2347
DOI: 10.1021/acs.accounts.6b00331 Acc. Chem. Res. 2016, 49, 2347−2354
Article
Accounts of Chemical Research
2. FLOW ELECTROCHEMICAL CYTOMETRY A hybrid microfluidic-capillary electrophoresis-electrochemical detection platform, flow electrochemical cytometry, was first developed to separate, lyse, and electrochemically measure the catecholamine contents of single vesicles (Figure 1).12 In this
important physiological roles: (i) to reduce the osmolality across the vesicular membrane by sequestration of catecholamine, theoretically estimated to be as high as 1500 mOsm, and (ii) to generate bioactive peptides, such as vasostatin, pancreastatin, and chromostatin.4 Neurotransmitters and neurohormones in the monoamine family, such as serotonin, histamine, dopamine, epinephrine, and norepinephrine, all readily undergo redox reactions. Specific monoamines, including dopamine, epinephrine, and norepinephrine, are found in high concentration (0.1−1.0 M) in adrenal chromaffin cells, and also the related rat adrenal pheochromocytoma cells (PC12 cells; an immortalized cell line stemming from the adrenal medulla of rats).2,5 Constant potential amperometry has been used extensively to study the release of these monoamines via exocytosis at single cells.6 Amperometric detection is beneficial owing to its high temporal resolution (95%. It has been reported that vesicular content released during exocytosis varies with experimental changes in stimulation such as vesicular pH manipulation, temperature change, osmotic pressure manipulation, etc.8 These results led to the hypothesis that normal exocytosis does not end in complete expulsion of the transmitter from each vesicle. To address this hypothesis, the vesicular catecholamine content was quantified with flow electrochemical cytometry, and was compared with the amount released during exocytosis.5 PC12 cells were chosen as a neuroendocrine secretory cell model as they have been widely used as a model cell for the study of secretion. Vesicles were isolated from PC12 cells with differential centrifugation and the isolation was confirmed successfully with Western blot by targeting synaptophysin, an integral membrane protein present on vesicles. Then the isolated vesicles were injected into the flow electrochemical cytometry system to quantify the vesicular 2348
DOI: 10.1021/acs.accounts.6b00331 Acc. Chem. Res. 2016, 49, 2347−2354
Article
Accounts of Chemical Research
Figure 2. Pharmacological treatment alters vesicular quantal size. (A) Representative normalized frequency histograms describing the distributions of vesicular catecholamine amounts quantified from stimulated exocytosis (A) and flow electrochemical cytometry (B) for reserpine-treated (red), untreated (blue), and L-DOPA-treated (black) cells. (C) Cumulative analysis for the average number of molecules of catecholamine quantified per vesicle from stimulated exocytosis of intact cells (striped) versus individual isolated vesicles (white) under pharmacological manipulation. Reproduced with permission from ref 5. Copyright 2010 American Chemical Society.
Figure 3. Pharmacological manipulation of striatal vesicle neurotransmitter content measured by flow electrochemical cytometry. (A) Representative current spikes for mice receiving reserpine treatment, untreated, or L-DOPA treatment. (B) Normalized frequency histograms for reserpine-treated (red) and L-DOPA-treated (blue) striatal vesicular dopamine content versus control (black). (C) Cumulative analysis of striatal vesicles from mice with respect to drug and time after treatment. Reproduced with permission from ref 11. Copyright 2013, by Nature.
vesicles isolated from mouse striatum.11 An average of 33 000 dopamine molecules were found stored in each vesicle in striatal tissue. This amount is much greater than that measured with release at brain tissue or for cultured neurons. Moreover, it is greater than the total amount of molecules released in repeated complex cases where the vesicle has been suggested to open and then close rapidly in a rapid flickering mechanism.15 In vivo administration of L-DOPA or reserpine alters the vesicular dopamine content markedly in a time-dependent manner (Figure 3), suggesting synaptic vesicle transmitter levels are not saturated in vivo. Furthermore, these data make it clear that transmitter stored in each vesicle can be regulated as well as the fraction released, making these aspects of cell function pharmacological targets. Additionally, in vivo administration of the psychostimulant amphetamine depletes vesicular dopamine by 50% after only 1 h. This measurement, difficult to make by any other current method, supports the weak-base hypothesis, which states that administration of a weak base drug causes vesicles to redistribute dopamine to the cytosol by collapsing the acidic vesicular pH gradient and/or interactions with the vesicular transporter.16
catecholamine content. Many current transients were recorded, with each transient corresponding to a single vesicle. Then the total vesicular catecholamine content was compared with the amount released during high K+ -stimulated exocytosis (detected by single cell amperometry with a 5-μm disk carbon fiber electrode) as shown in Figure 2. The amount released during exocytosis was significantly less than the total vesicular content measured, revealing that exocytosis does not result in complete expulsion of the transmitters in a vesicle. L-DOPA (L3,4-dihydroxyphenylalanine) is a biosynthetic precursor to dopamine and a drug known to increase dopamine levels.13 Reserpine is a drug known to block the transport of dopamine into vesicles, thus allowing it to leak out.14 Treatment with LDOPA increases the vesicular catecholamine content, whereas reserpine treatment decreases it when monitored by electrochemical cytometry. Interestingly, the fraction of catecholamine released during exocytosis is kept in the same range (∼40%) for untreated, L-DOPA treated, and reserpine treated cells. A comparison of the vesicular catecholamine content and vesicle substructure size measured by dynamic light scattering and transmission electron microscopy suggest that the catecholamine reserves are nearly equally located in both the protein dense core and the solution surrounding the dense core of the vesicle. The flow electrochemical cytometry system has also been used to measure the dopamine content in individual synaptic
3. IMPACT ELECTROCHEMICAL CYTOMETRY FOR SOFT NANOPARTICLES A new approach, impact electrochemical cytometry (Figure 4) can also be used to investigate the electroactive content of soft 2349
DOI: 10.1021/acs.accounts.6b00331 Acc. Chem. Res. 2016, 49, 2347−2354
Article
Accounts of Chemical Research
probability of electroporation. It is also likely that the proteins must diffuse away from the section of the vesicle in contact with the electrode in order to allow the membrane to approach sufficiently close to the surface to undergo electroporation. The dynamics of these events is also affected for these three different systems (Figure 5) with the clean liposomes having
Figure 4. Schematic diagram of a vesicle sticking to the electrode and then opening allowing species inside to be measured via electrolysis at the electrode. The reaction shown is norepinephrine oxidation. Reproduced with permission from ref 9. Copyright 2015 American Chemical Society.
Figure 5. Duration of events related to control liposomes (without peptide), liposomes with peptide and chromaffin vesicles; ***p < 0.001 using a two tailed paired Student’s t test. Trace acquisition was as follows: control liposomes: 3 sample preparations, 16 traces in total; peptide liposomes: 4 sample preparations, 24 traces in total and chromaf f in vesicles: 6 vesicle isolations, 18 traces in total. Reproduced with permission from ref 17. Copyright 2016, by RSC.
nanoparticles.9 Here, a 33 μm disk carbon fiber electrode is either dipped in a concentrated suspension of isolated vesicles for 20 min before recording signals in a bulk buffer or maintained in a relatively diluted vesicle suspension for direct measurement. In both procedures, vesicles diffuse to the electrode surface and then either bounce or adsorb to it. Owing to the high stability of large dense core vesicles isolated from chromaffin cells, a rather large population of vesicles can adsorb to the electrode surface prior to opening. These vesicles subsequently rupture via an electroporation mechanism to release their content in very close vicinity to the electrode surface resulting in complete oxidation.17 Electroactive species in the vesicle are oxidized (or reduced) generating an amperometric current transient, allowing quantification of vesicular content, again using Faraday’s law, and providing kinetic information about the vesicle opening process. This approach has been used to show that the number of events observed is correlated to the applied potential, with more events at higher potential.17 This suggests that the mechanism of vesicle rupture involves electroporation to form a pore between the interior of the vesicle and the electrode. With a membrane thickness of 5 nm and an applied potential of 700 mV the electric field across the membrane is in the range of 1.4 × 106 V cm−1 and sufficient enough for electroporation.18 The applied potential is related to the electrostatic energy as CV2/2, where C is the capacitance of the membrane and V is the potential difference across the membrane. As a consequence of the increased energy, the probability for pore formation is increased, allowing for a larger fraction of the vesicles adsorbed on the electrode surface to rupture and release their contents. Chromaffin vesicles are very complex structures with, in addition to the monoamines and protein dense core, a wide range of transmembrane proteins decorating the vesicular exterior. In order to study the vesicle rupture and opening process, less complex systems like clean liposomes (no added peptide) and liposomes decorated with octaarginine peptides have been used. Clean liposomes show about 20 times as many events for the same particle number as chromaffin cell vesicles leading to the suggestion that the protein decorating the surface is a key player in the rate of vesicle opening.17 Proteins in the membrane of the chromaffin vesicles appear to act as a physical barrier reducing the electrical field across the membrane leading to a decreased electrostatic energy and consequently reduce the
the fastest response time (event half width,
