Fast-Scan Cyclic Voltammetry for Ex Vivo Recording of Dopamine Release

Summary

Fast-scan cyclic voltammetry (FCV) measures rapid dopamine dynamics with sub-second resolution using carbon-fibre microelectrodes. This protocol describes FCV in acute mouse brain slices, including electrode fabrication, slice preparation, calibration, and stimulation-based assays to examine how cholinergic interneurons regulate striatal dopamine release under physiological conditions.

Abstract

Fast-Scan Cyclic Voltammetry (FCV) is an electrochemical technique that enables real-time detection of oxidisable neurotransmitters, including dopamine (DA), norepinephrine, and serotonin, with sub-second temporal resolution. By applying a rapidly scanning triangular voltage waveform to a carbon-fibre microelectrode, FCV allows highly sensitive measurement of rapid, transient changes in extracellular neurotransmitter concentrations with excellent temporal precision. This makes FCV particularly well-suited for recording fast neuromodulatory signalling events. This article describes the application of FCV to measure evoked DA release in ex vivo mouse brain slices. The protocol outlines essential steps for carbon-fibre microelectrode fabrication, acute brain slice preparation, electrode conditioning, data acquisition, and post-hoc calibration to convert current signals into absolute DA concentrations. Using the selective β2-containing nicotinic acetylcholine receptor (nAChR) antagonist dihydro-β-erythroidine (DHβE), we demonstrate that nAChR activity has a powerful modulatory influence on striatal DA release. Together, this work highlights FCV as a powerful approach for investigating monoamine transmission across brain regions.

Introduction

Fast-scan cyclic voltammetry (FCV) is an electrochemical technique developed to monitor rapid fluctuations in electroactive neurotransmitters¹. Unlike traditional cyclic voltammetry, which uses slow scan rates¹, FCV applies a triangular potential waveform at a much faster scan rate (typically 400 V/s) through a micrometre-scale carbon-fibre microelectrode, enabling subsecond measurements of neurotransmitter concentration². These high scan rates generate a large but stable background charging current that can be digitally subtracted to isolate the faradaic currents produced by oxidation and reduction reactions of neurotransmitters^2,3.

FCV enables real-time measurement of extracellular neurotransmitter concentrations in specific brain regions, providing high temporal resolution and moderate chemical selectivity⁴. These features make FCV well-suited for studying rapid neurochemical signalling in both in vivo and ex vivo preparations, including freely moving or anaesthetised animals and acute brain slices^5,6,7. This technique offers several advantages: (1) controlled stimulation protocols can be used to evoke defined dopaminergic responses; (2) the small diameter of carbon-fibre microelectrodes minimises tissue damage and allows precise spatial recordings; (3) FCV is highly compatible with pharmacological manipulations, enabling efficient investigation of monoamine release and uptake kinetics; and (4) the combination of chemical selectivity and high temporal resolution provides improved detection compared with other electrochemical methods, such as amperometry, and sampling approaches like microdialysis, which often lack the speed to capture rapid, transient neurotransmitter events or face challenges in identifying and quantifying specific molecules⁵.

Dopamine (DA) is a key neuromodulator involved in reward, motivation, motor control, and multiple aspects of learning and memory, and it is characterised by rapid and transient release dynamics⁸. In FCV, a triangular voltage waveform, typically sweeping from -0.4 V to +1.2 V, is repeatedly applied to a carbon-fibre microelectrode. During the anodic (upward) sweep, DA near the electrode surface is oxidised at approximately +0.6 V, releasing two electrons and forming dopamine-o-quinone. During the cathodic (downward) sweep, this compound is reduced back to DA at around -0.2 V, taking up two electrons⁹. The resulting current-voltage signature enables precise detection and quantification of fast DA release and clearance with high spatial and temporal resolution, revealing second-to-second fluctuations in extracellular DA levels. In our experiments, FCV recordings were collected at 10 Hz for 15-30 s, which was sufficient to capture DA release evoked by electrical stimulation.

Within the striatum, DA release is tightly regulated by local cholinergic interneurons (ChIs). Although ChIs constitute only ~1%-2% of striatal neurons, they arborise extensively and have strong control over dopaminergic axons through axo-axonic interactions^10,11. This regulation is likely to play a critical role in striatal learning. During learning, midbrain DA neurons exhibit phasic firing in response to rewards or reward-predicting cues¹², whereas ChIs display a highly synchronised, multiphasic excitation-pause-rebound firing pattern¹³. The temporal coincidence between the ChI pause and phasic DA activity further underscores the importance of ChI-mediated regulation of DA release via nicotinic acetylcholine receptors (nAChRs)^14,15. Beyond regulating DA release, this temporal coincidence has been shown to be necessary for the induction of learning-related synaptic plasticity in the striatum¹⁶, indicating the importance of clarifying the interactions between ChIs and DA signalling within this circuit.

ChIs influence DA release through at least two distinct mechanisms. First, synchronous activation of ChIs can evoke rapid, short-latency DA release that occurs independently of dopaminergic somatic firing via activation of nAChRs on DA axons^17,18,19. This form of axonal DA release is likely mediated by the generation of ectopic action potentials within DA axons¹⁷. Second, ChI activation can profoundly suppress subsequent DA release by preventing depolarisation of DA axons²⁰. Notably, this suppression occurs with very short latency and can be induced by relatively low levels of ChI activity that do not themselves elicit detectable DA release, suggesting that activation of nAChRs is more effective at suppressing ongoing DA release than at triggering DA release. Therefore, these factors suggest that ChIs may exert an overall suppressive influence on DA release under physiological conditions. However, at a finer spatial scale, ChIs may locally facilitate DA axonal release.

Collectively, these findings highlight a finely tuned regulatory role for ChIs in controlling striatal DA signalling²⁰. FCV provides a powerful approach for dissecting these Chl-DA interactions, as its high temporal resolution enables precise measurement of both the facilitatory and suppressive effects of ChI activity on DA release.

This article outlines the essential procedures for fabricating carbon-fibre microelectrodes and measuring evoked extracellular DA concentrations using FCV in ex vivo preparations.

Protocol

All animal procedures were approved by the University of Exeter Animal Welfare and Ethical Review Board and conducted under Home Office License [PP262252]. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of carbon-fibre microelectrodes

1. Fill a clean glass container with acetone. Submerge the glass capillaries fully in the acetone, ensuring that each capillary is filled.
2. Using tweezers, insert a single carbon fibre into an acetone-filled capillary. Remove the capillaries from the acetone and allow them to air-dry completely.
3. Load the dried capillary tube into a vertical microelectrode puller, positioning the midpoint of the tube in line with the heating filament.
4. Activate the puller and set the heater level to 58.1 to produce two carbon-fibre electrodes from a single capillary tube. Collect the pulled electrodes for trimming.
   NOTE: There are no universal settings for heating temperature or pulling force. These parameters vary across instruments and must be optimised through trial and error for each laboratory setup.
5. Place the pulled electrode under a microscope equipped with a 10× reticle. Using a scalpel, trim the fibre tip to a length of ~100 µm (Figure 1A).
6. Strip approximately 1 cm of insulation from each end of a conductive wire. Apply a thin layer of conductive silver ink to one exposed end.
7. Insert the silver-coated end of the wire into the back end of the carbon-fibre electrode, ensuring firm contact between the wire and the fibre. Seal the connection with super glue and allow it to dry completely.

2. Preparation of solutions

1. Prepare the sucrose-based cutting solution.
   1. To prepare 1 L of sucrose-based cutting solution (pH 7.4, ~300 mOsm/L), dissolve the following components (in mM): 85 NaCl, 25 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 7 MgCl₂, 10 glucose, and 65 sucrose.
   2. Transfer 200 mL of the sucrose-based cutting solution into a clean bottle. Add 200 µL of 1 M CaCl₂·H₂O to reach a final CaCl₂ concentration of 1 mM. Bubble the solution with a gas mixture of 95% O₂ / 5% CO₂ for at least 30 min before use.
2. Prepare artificial cerebrospinal fluid (aCSF, pH 7.4, 300 mOsm/L) for recording.
   1. Prepare a 10× aCSF stock solution by dissolving the following in 1 L of distilled water: 1300 mM NaCl, 250 mM NaHCO₃, 25 mM KCl, and 12.5 mM NaH₂PO₄. Store the stock solution at 4 °C.
   2. To prepare 1× aCSF, combine 100 mL of the 10× aCSF stock solution with 52 mL of 0.5 M NaHCO₃. Dilute the mixture to 1 L with distilled water. Add 2 mL of 1 M CaCl₂·H₂O to obtain a final concentration of 2 mM CaCl₂. Bubble the solution with 95% O₂/5% CO₂ for at least 30 min before use.
3. Prepare 2.5 mM DA standard stock solution for calibration
   1. Prepare 0.1 mM perchloric acid by diluting 20 µL of 0.1 M perchloric acid into 20 mL of distilled water. Mix thoroughly to ensure complete dilution.
   2. Weigh 9.48 mg of DA hydrochloride powder and dissolve it in the 0.1 mM perchloric acid prepared above to obtain a final concentration of 2.5 mM. Store the solution at 4 °C and protect it from light to prevent oxidation.

3. Brain slice preparation

1. Euthanize the animals, then rapidly extract the brain and immediately transfer it into ice-cold sucrose-based cutting solution.
2. Mount the brain onto the vibratome platform and cut 300 µm coronal slices between +1.5 mm and +0.5 mm relative to bregma at a cutting speed of 0.34 mm/s.
3. Transfer the slices to a recovery chamber containing aCSF maintained at 32 °C. Incubate the slices for 30-40 min.
4. After recovery, maintain the slices at room temperature in a continuously oxygenated aCSF until further use.

4. Rig setup and electrode conditioning

1. Switch on the perfusion system. Perfuse the entire rig with distilled water for at least 10 min. Turn on the heating system and set the temperature to 32 °C.
   NOTE: Ensure that the temperature sensor is fully immersed in the solution before activating the heater.
2. Turn on the stimulus generator software, camera, micromanipulator, and microscope background illumination.
3. Open the HDCV software for FCV data acquisition. Set the voltage parameters to a triangular waveform sweeping from -0.4 V to +1.2 V at a scan rate of 400 V/s, applied at 10 Hz.
4. Place the brain slice in the centre of the slice chamber and secure it using a harp.
5. Using the micromanipulator, bring the electrode tip into the field of view of the microscope.
6. Check the electrode tip under the microscope to confirm that it is intact and not broken.
7. Start data collection and check the current-voltage plot.
8. Condition the new electrode by placing it in cortical tissue and running a triangular waveform cycling for at least 30 min before recording.
   NOTE: A proper cyclic voltammogram should display a characteristic charging curve (Figure 1B). Replace the electrode if the voltammogram is distorted (Figure 1C-E) or if the background current is unstable. If instability persists, check the grounding connection.

5. Measurement of extracellular DA concentration

1. Using the micromanipulator, move the recording electrode from its conditioning site to the target recording location within the brain slice.
2. Position a concentric bipolar stimulating electrode at the desired recording site. Place the recording electrode approximately 50 µm from the stimulation electrode (Figure 2A,B).
3. Deliver single-pulse electrical stimulation at 0.65 mA with a pulse width of 200 µs and an inter-stimulus interval of 150 s. Verify that the oxidation and reduction peaks occur at the expected voltages (Figure 2C) and that the DA transient shows typical release and clearance kinetics (Figure 2D).
4. Record 10 baseline DA signals and review the final three baseline traces. Confirm that variation across these signals is less than 5%.
   NOTE: If signal amplitude is low or continues to decline, check the carbogen supply, temperature stability, and the osmolarity and pH of the aCSF.
5. Deliver stimulation pulses using a pseudorandomised sequence that includes both single-pulse (1p) and four-pulse (4p, 100 Hz) paradigms.
   NOTE: The 4p 100 Hz stimulation mimics the natural high-frequency firing of DA neurons¹¹ and is commonly used to assess the maximal magnitude of ChI-dependent depression, which occurs approximately 7-8 ms after ChI activation²⁰. Alternate stimulation patterns throughout the recording to minimise sequence bias.
6. Dissolve the desired pharmacological agent in aCSF and bubble the solution with 95% O₂/5% CO₂ for at least 10 min.
7. Apply the drug to the bath chamber and incubate until the full pharmacological effect is reached (e.g., 10-15 min for DHβE). Repeat the stimulation and recording procedures under drug-treated conditions.

6. Calibration

1. Prepare a 2 µM DA calibration solution by diluting 20 µL of the 2.5 mM DA stock into 25 mL of oxygenated aCSF.
2. Position the electrode near the inlet. Adjust the microscope focus to visualise the upper and lower edges of the inlet clearly. Refocus on the centre of the inlet tube and adjust the micromanipulator (raise or lower) until the electrode tip is clearly visible.
3. Perfuse the electrode with oxygenated aCSF at a rapid flow rate for approximately 10 s to wash the electrode surface. While observing the switch under the microscope, change the perfusion line from aCSF to the DA calibration solution.
4. Maintain perfusion with the DA solution for 30-60 s. Switch the tubing back to aCSF and continue perfusion to wash out the DA. Repeat the entire calibration procedure three times.
5. Calculate the calibration factor (nA/µM) using the average current response (ΔI) from the three DA applications (Figure 2E). For each application, compute ΔI using: ΔI = I_DApeak− I_DAbase. Average the ΔI values and divide by the known DA concentration (2 µM) to obtain the electrode’s calibration factor.
6. Label the electrode with the experiment date and save all calibration data files for future reference.
   NOTE: Each recording electrode is calibrated at the end of the experimental day and is not reused for subsequent recordings to ensure optimal sensitivity and accuracy.

Results

DA release amplitudes were quantified from background-subtracted FCV recordings. For each trial, the background signal was generated by averaging five stable pre-stimulation voltammograms, enabling clear isolation of the oxidation peak. DA release was measured as the peak oxidation current at approximately +0.6 V. At the end of each experiment, electrodes were calibrated, and peak oxidation currents were converted to DA concentrations using the electrode-specific sensitivity factor (nA/µM). To facilitate comparisons acro...

Discussion

This protocol describes the fundamental procedures for measuring evoked DA release using FCV in acute mouse brain slices and provides a comprehensive guide to the essential workflow. In addition to DA-specific applications, FCV can also be used to detect other neurotransmitters, such as noradrenaline and serotonin, in both ex vivo and in vivo preparations^21,22,23. Here, we highlight the key components of the me...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetone	Sigma-Aldrich	534064-500ml
Calcium Chloride	Sigma-Aldrich	21115-100ml	1M solution
Carbon Fiber	Goodfellow Cambridge Limited	LS596012 APB
Concentric Bipolar Electrode	FHC	300197	Stimulus Electrode
Dihydro-β-erythroidine hydrobromide	Tocris Biosciences	12A/295293
Dopamine HCl	apexbt	B1482
Fast Scan Cyclic Voltammetry Potentiostat System	WaveNeuro	AF01FSCV1
Glass Capillaries	Harvard apparatus	GC200F-10
Glucose	Sigma-Aldrich	SLCQ7550
Halogen Power Supply	Olympus	TH4
Isolated Current Stimulator	Digitimer	DS3
Magnesium chloride hexahyrate	Sigma-Aldrich	M2670
Multicore Cable	Multicomp Pro	3372784
Perchloric acid	Fisher science	UN2920	0.1M in glacial acetic acid
Peristaltic Pumps	Gilson	MINIPLUS 3
Potassium chloride	Sigma-Aldrich	P9541
Prime BSI Express	Teledyne	01-PRIME-BSI-EXP
Silver Conductive Paint	RS Components	RS 186-3600
Silver/Silver Chloride Reference	World Precision Instruments	0226E	Reference Electrode
SliceScope	Scientifica	Pro 2000
Sodium bicarbonate	Sigma-Aldrich	S5761
Sodium chloride	Fisher science	2402665
Sodium phosphate monobasic	Sigma-Aldrich	S5011
Stimulus Generator	Multichannel systems	STG5
Sucrose	Millipore	84100
Vertical microelectrode puller	Narishige	PC-100