Analyzing Fusion Pore Dynamics and Counting the Number of Ace-tylcholine Molecules Released by Exocytosis

: Acetylcholine (ACh) is a critical neurotransmitter influencing various neurophysiological functions. Despite its significance, there is a lack of quantitative methods with adequate spatiotemporal resolution for recording single exocytotic efflux of ACh. In this study, we present an ultrafast amperometric ACh biosensor enabling electrochemical recording that captures spontaneous bursts of single presynaptic exocytosis events at axon terminals of cholinergic cells with sub-millisecond temporal resolution. Characterization of the recorded amperometric time trace revealed seven distinct current spike types, each displaying variations in both spike shape, time scale and quantities of ACh released through the synaptic vesicle fusion pore. This observation suggests the presence

ABSTRACT: Acetylcholine (ACh) is a critical neurotransmitter influencing various neurophysiological functions.Despite its significance, there is a lack of quantitative methods with adequate spatiotemporal resolution for recording single exocytotic efflux of ACh.In this study, we present an ultrafast amperometric ACh biosensor enabling electrochemical recording that captures spontaneous bursts of single presynaptic exocytosis events at axon terminals of cholinergic cells with sub-millisecond temporal resolution.Characterization of the recorded amperometric time trace revealed seven distinct current spike types, each displaying variations in both spike shape, time scale and quantities of ACh released through the synaptic vesicle fusion pore.This observation suggests the presence of multiple exocytosis modes at these cells.Quantifying the absolute number of ACh molecules released at single exocytosis events was achieved through sensor calibration using electroanalytical measurements of synthetic lipid vesicles containing varying concentrations of ACh.Notably, the largest quantal release was estimated at approximately 8000 ACh molecules, likely representing full exocytosis, while the fractional release of roughly 5000 ACh molecules correspond to a partial exocytosis mode.Following a local administration of bafilomycin A1, a V-ATPase inhibitor, the cholinergic cells exhibited both a higher frequency and larger quantity of ACh released during exocytosis events.Hence, this ACh sensor introduces means to monitor minute amounts of ACh and investigate regulatory mechanisms at single-cell level, which is vital for understanding healthy brain function, pathologies, and optimizing drug treatment for disorders.
Neuronal communication occurs through the rapid, calciumtriggered release of neurotransmitters from synaptic vesicles fusing with the cell plasma membrane. 1This process, known as exocytosis, happens in less than a millisecond, 2 allowing neurotransmitters released to bind to receptors at neighboring neurons and transmit chemical signals. 1 The amount and frequency of neurotransmitter released are important factors in determining the communication strength between neurons and in shaping synaptic plasticity.This process underlies fundamental brain functions such as learning and memory, and is also involved in the pathophysiology of brain disorders such as drug abuse and addiction, which are not yet fully understood. 3,4Acetylcholine (ACh) is a key excitatory neurotransmitter in the central and peripheral nervous systems (CNS and PNS), 5 essential for regulating heart rate, controlling muscle contraction, and contributing to cognitive function. 6,5It also serves as a neuromodulator of glutamatergic and GABAergic synapses in the CNS. 7The impairment of the cholinergic system has been associated with several neurological and psychiatric disorders, such as Alzheimer's disease and schizophrenia. 8Investigations of cholinergic neurotransmission and its role in complex cognitive behavior, have led to the development of various analytical techniques for studying ACh signaling, such as in vivo microdialysis, capillary electrophoresis, mass spectrometry, neuroimaging, and photoelectrochemical biosensing. 9,10,11However, for an in depth understanding of the regulatory aspects of ACh neurotransmission at synapses, methods that spatially and temporally can capture the dynamics of rapid synaptic vesicle ACh release during neuronal activity are needed.Synaptic neurotransmission can be studied using electrochemical methods where carbon fiber amperometry is commonly used due to its straightforward application and superior temporal resolution (µseconds).By placing carbon fiber micro/nanoelectrodes at release sites of single cells, redox currents in response to detection of neurotransmitters released from single exocytosis events can be recorded.This method enables the recording of vesicle fusion pore-controlled release dynamics of single synaptic exocytosis events that occur on sub-millisecond timescale. 12Further, amperometry is quantitative and the number of molecules released during exocytosis can be determined, resulting in a measure of synaptic signaling strength.The integrated charge (Q) from individual current spike detected, can be converted into the number of molecules released using Faraday's law. 12However, its applicability is limited to electroactive neurotransmitters, such as catecholamines and serotonin, which can be readily oxidized at the electrode surface.For non-electroactive neurotransmitters like ACh and glutamate, redox reactions at the carbon surface are not possible.To address this, a chemically selective biosensor can be created by modifying the electrode surface with enzymes capable of converting non-electroactive neurotransmitters into detectable reporting molecules, usually hydrogen peroxide (H2O2).While these biosensors are sensitive and selective, their detection capabilities have remained limited to sub-second time resolutions, which is too slow for monitoring individual exocytosis events. 13Insufficient technology for direct quantitative measurements of ACh then has led to varying estimates of ACh vesicle quantal size, ranging from 400 to 22,000 ACh molecules, depending on the biological model system and analysis techniques used. 14,15,16,17,18,19,20,21To overcome this challenge, our lab introduced a novel biosensor design concept that improves sensor speed by limiting enzyme coatings to a molecular monolayer.This approach minimizes diffusion distances for enzymatic electroactive reporter molecules to reach the electrode surface for detection. 22Compared to traditional sensors with thick enzyme layers, our minimal enzyme-coating concept has reduced response times by up to 3 orders of magnitude.With a 33-µm carbon fiber biosensor, we have captured ACh release from artificial cells in tens of milliseconds, 22 and glutamate from exocytotic release in brain tissue and isolated synaptic vesicles in sub-milliseconds. 23,24n this work, we tailored our ultrafast sensor concept for single cell ACh presynaptic measurements by immobilizing the sequential enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) onto a 5 μm carbon fiber disc electrode surface (Figure 1A), providing dimensions for precise positioning against single cell structures.To functionalize the carbon microelectrode surface, maximizing its sensing surface area, enhancing detection efficiency of H2O2 and providing a scaffold for the ultrathin enzyme coating, a dense layer of gold nanoparticle (AuNP) hemispheres, approximately 80 nm in diameter, was electrodeposited, creating a surface topology resembling to cauliflower (Figure 1D, Figure S2).The enzymes AChE and ChO were then immobilized onto the AuNP-modified sensor surface at a molar ratio of 1:10, based on a previous study optimizing ACh sequential enzymatic catalysis (Figure 1A). 25 The ACh biosensor created was then placed in contact to axon terminals of differentiated cholinergic human SH-SY5Y neuroblastoma cells (Figure 1B,F and Figure S3).In Figure 1C, and detailed in Table S1, when a constant potential of -0.5V versus a Ag/AgCl reference electrode was applied to the ACh sensor surface, the sensor successfully captured spontaneous bursts of isolated reduction current transients corresponding to individual ACh exocytotic events at the submillisecond timescale (n = 18 cells).The average frequency of ACh release was measured at 28 ± 5 Hz (n = 16 cells), with the error representing the standard error of the mean (SEM) and the average current spike from these recordings shows the typical shape characteristics of fusion pore regulated exocytosis with a rapid Trise and slower Tfall (Figure 1E).To demonstrate how the ACh biosensor can be used to investigate the drug effect on ACh signaling, single cholinergic cells were exposed to 0.1 μM bafilomycin A1 (Figure 1G,H), a V-ATPase inhibitor known to also activate release of intracellular calcium stores, potentially affecting fusion pore dynamics and exocytosis activity. 27By recording ACh release from a single release site before and after a 10-min local administration of the drug using a glass microinjection pipette, an increase in both the exocytosis activity (170%) and quantal release (120%) was observed (Figure 1I, Figure S7 and Table S2).This finding is consistent with a previous study on catecholamine signaling in chromaffin cells and demonstrates the applicability of this technology for presynaptic studies. 28he high temporal resolution of our recordings (50 kHz) enabled detection of a diversity of shapes and time courses for individual current spikes.This spike heterogeneity indicates that the fusion pore is highly regulated by factors controlling its opening and closing and leads to presynaptic tuning of ACh release in these cells.Figure 2D presents the spike shapes identified and shows that the highest abundance of spikes corresponds to "sharp spikes" (42%).Many of these spike types have been previously identified with comparable prevalence in brain tissue recordings of octopamine in Drosophila melanogaster and glutamate in rodents. 29,30In these recordings, a novel category of sharp spike was detected, adding to the six previously observed spike types associated with partial, full and multi-release in glutamatergic exocytosis. 23This spike, predominant at 9%, exhibits a sharp simple shape (depicted in Figure 2D).Compared to the other current spike types, it has approximately twice the current amplitude (Imax) and result in a similar total charge detected as spikes representing full exocytosis mode (Figure S5).Hence, we classify these spikes as 'full sharp' spikes.To accurately quantify the number of ACh molecules by exocytosis release, an appropriate calibration of the sensor signal is needed.Here we used a liposome-based calibration strategy, pioneered by our lab for quantifying glutamate. 31This method involves immersing the ACh sensor into a solution of large unilamellar liposomes (LUVs) preloaded with ACh and performing amperometric recording of single LUV content. 32,33y applying a potential to the biosensor, individual LUVs in contact with the sensor surface stochastically rupture and the released ACh is detected as current spikes.Figure 2A and B display a typical redox current time trace, detecting ACh release from single LUVs pre-filled with a 400 mM ACh solution, demonstrating detection of single current spikes on sub-millisecond timescale (Figure S8B, Table S3).Extruded LUVs (~180 nm in diameter) pre-filled with various ACh concentrations (200 mM to 600 mM) underwent electroanalysis using the ACh sensor.
The integrated total charge (Q) of individual redox current spikes (Figure 2B, Table S3) was used for calculating the average charge density (Q/LUV size).The linear relationship when plotting the charge density against the ACh concentration encapsulated within the LUVs establishes a calibration curve for cellular recordings (Figure 2C).The absolute quantification of ACh released at exocytosis was then performed for each spike type using the calibration curve.Figure 2E, illustrates that the analysis indicates both complex plateau-shaped and full sharp events release an average of 8400 ± 320 ACh molecules (mean ± SEM), implying full exocytosis and complete release of vesicular ACh content.This estimation align with the consensus of 5,000 to 10,000 ACh molecules per vesicle. 34In contrast, the most prevalent, 'sharp spikes', release an average of 4700 ± 160 ACh molecules (mean ± SEM).This indicates partial exocytosis release, consistent with earlier glutamate measurements for this spike type. 30,35 igure 2F shows that pooling all release events detected into a histogram, a LogNormal equation fit estimates an average release of 5800 ACh molecules across all spike modes.Assuming an internal vesicle solution volume of 20 zL, 36 we estimate the original vesicular ACh concentration to be 0.7 ± 0.03 M, which is similar to findings for storage of glutamate and catecholamine in secretory vesicles. 35,37,38n summary, we present the development of an ACh biosensor and its application for monitoring the temporal dynamics of quantal release during exocytosis from presynaptic sites at human differentiated cholinergic cells with sub-millisecond precision.The high temporal resolution of the sensor facilitated the study of drug effects on exocytosis activity and quantal release, as well as the identification of various dynamic exocytosis modes.This ultrafast technology offers a novel approach to studying the regulatory aspects of fusion pore-controlled release and provides insight into the ACh system involved in both healthy neurotransmission and neurological disorders.

Figure 1 .
Figure 1.(A) Detection principle for the ultrafast ACh biosensor, modified with AuNPs (red hemispheres) and the enzymes AChE (blue) and ChO (yellow).Schematic not to scale.(B) Experimental setup with placing an ACh biosensor at an axon terminal of a differentiated cholinergic SH-SY5Y cell, opposing a glass pipette delivering stimulants or drugs.Scale bar is 10 μm.(C) A 50 kHz amperometry recorded current time trace of ACh exocytosis activity at an axon terminal.(D) SEM image of a 5-μm carbon electrode coated with a dense layer of ~80 nm AuNPs.Scale bar: 1 μm.(E) An average amperometric current spike representing ACh release from single exocytosis events (n=162).(F) TEM image of a cholinergic SH-SY5Y cell axon displaying the presence of synaptic vesicles.Scale bar: 0.3 μm.(G) Exocytosis amperometry recording performed as control experiment the presence of 0.002% DMSO (gray) and (H) following a 10-minute local administration of 0.1 μM bafilomycin A1 (red) using the same ACh biosensor at a precise location of a target cell.(I) Comparison of drug effect to single exocytosis events regarding ACh current spike halftime (T1/2), maximum amplitude (Imax), total charge (Q) and frequency (f) (average of means ± SEM) with control.Two-tailed paired Mann-Whitney test was used, ****p < 0.0001.

Figure 2 .
Figure 2. (A) Amperometric trace of the reduction current transients due to the stochastic rupture of LUVs preloaded with 400 mM ACh and detection of ACh release at the sensor surface upon applying -0.5 V vs a Ag/AgCl reference electrode.(B) Each current transient from single LUV rupture provides kinetic and quantitative information.(C) Calibration curve plotting the mean charge density from LUV electroanalysis vs LUV preloaded ACh concentration (n = 4-5 LUV samples, 600-2000 spikes/ACh concentration), showing the average value ± standard error of the mean (SEM).(D) Relating the ACh quantal release from single exocytosis events to the various detected current spike shapes and their detected abundance (%).(E) Comparison of ACh quantal release vs different category of current spike shapes (average of means ± SEM) using two-tailed unpaired Student's t test, ****p < 0.0001.(F) LogNormal histogram (bin size 1, R 2 = 0.98) displays the cubic root transformed distribution of ACh molecules released from single exocytosis events.D) -F) Data is collected from 18 cells, and 2945 amperometric current spikes recorded.