Name: Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry (Video) | JoVE Journal
Uploaded: 2012-01-12T13:00:00.000+00:00
Duration: 8 min 49 s
Description: Learn fast-scan cyclic voltammetry for measuring presynaptic dopamine dynamics in striatal brain slices. This method enables real-time observation of dopamine release and uptake in neuroscience research.

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Funding provided by the National Institute on Alcohol Abuse and Alcoholism (NIAAA; AA-016967 and AA016967-01S1; TAM), Wayne State University start up funds, and Wayne State University Research Grant Program. The content is solely the responsibility of the authors and does not represent the official views of NIAAA or the National Institutes of Health.

1. Experimental essentials
Electrode Fabrication
<ul type="circle">
 <li>There are numerous carbon fiber microelectrodes fabrication methods since most are made in-house. Typically what dictates the electrode fabrication details is the electrochemical technique that is applied to the electrode (e.g. amperometry vs. FSCV). For FSCV, microelectrodes can be made in-house using the following three-step procedure. For a more complete description of carbon fiber electrode fabrication, see a recent JOVE article 1. However, note that the electrodes described below are cylindrical carbon fiber microelectrodes, which require fewer steps to fabricate versus the amperometric carbon fiber microelectrodes from the above-mentioned protocol. This simplified protocol does not require boiling the carbon fiber in acetone, fire-polishing the glass capillaries, or using epoxy to seal the glass-fiber junction.</li>
 <li>Using vacuum suction aspirate a carbon fiber (diameter 7 μm; Goodfellow Oakdale, PA) into a borosilicate glass capillary with microfilament (length 10 cm, o.d. 1.2 mm, i.d. 0.68 mm; A-M systems, Carlsborg, WA).</li>
 <li>Place the threaded capillary into the electrode puller (Narishige, Tokyo, Japan) where the capillary is pulled in half. Output settings for the electrode puller do vary from lab to lab. For reference, our output settings for the puller are 90.7 main magnet, 23.2 sub-magnet, and 53.4 for the heater. The output settings should be empirically refined to generate a glass taper that is approximately 4.4 mm in length, with a tight seal around the carbon fiber.</li>
 <li>Under a microscope (Olympus, Tokyo, Japan), trim the carbon fiber (using a scalpel blade) extending from the glass tip allowing approximately 50-200 μm of the carbon fiber to protrude from the tightly sealed interface.</li>
</ul>
Solution Preparation
Three types of artificial cerebrospinal fluid (aCSF) solutions need to be prepared in advance, all in ultrapure (18 MΩ cm) water.
<ul type="circle">
 <li>Sucrose-aCSF can be prepared at least one day before the slicing. The sucrose-aCSF buffer consists of (in mM): 180 sucrose, 30 NaCl, 4.5 KCl, 1.0 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose (pH 7.4) and should be oxygenated using 95% O2/5% CO2 for 15 minutes 2. If prepared ahead of time, the solution can be kept refrigerated at 4 °C for up to 1 week.</li>
 <li>ACSF solution for voltammetric recordings should be prepared the day of the experiment. The aCSF solution consists of (in mM): 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, 11 D-glucose, 0.4 ascorbic acid (pH 7.4). During the course of the experiment, oxygenate by bubbling with 95% O2/5% CO2 at room temperature.</li>
 <li>Modified-aCSF solution for electrode calibrations contains (in mM): 2.5 KCl, 126 NaCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.2 MgCl2, and 25 NaHCO3 (pH 7.4) and can be kept for up to one week, without refrigeration or oxygenation.</li>
</ul>
Electrode Calibration
<ul type="circle">
 <li>Electrode viability and sensitivity is determined by pre- and post-calibration, respectively, using a flow "t-cell" containing 3 ports with a sealed reference electrode (Ag/AgCl). The fabricated microelectrode is lowered into the flow "t-cell". The inlet port is connected to a syringe pump, allowing for a continuous flow of modified-aCSF at a flow rate of 2 mL/min. The third port of the "t-cell" is connected to a syringe filled with 3 μM dopamine, made in modified-aCSF.</li>
 <li>To pre-calibrate, allow modified aCSF solution to flow for about 3 - 7 seconds, and then manually inject 1-2 mL of the 3 μM dopamine standard. For each electrode to be pre-calibrated, repeat the calibration at least 3 times and average the maximal currents obtained from each.</li>
 <li>Immediately after the slice voltammetry experiment (see section 3), the electrode is post-calibrated in the same manner as described above.</li>
 <li>The calibration factor (later used during data analysis) is determined by dividing the average dopamine oxidation current (nA) by the concentration of the dopamine standard. For example, the current response is divided by 3 when using a 3 μM dopamine standard.</li>
</ul>
2. Slice preparation
<ol>
 <li>Pour 10 mL of sucrose aCSF into a small beaker and place in an ice bucket. Additionally, place instant adhesive (Loctite 404) in ice bucket, within reach.</li>
 <li>Prepare a razor blade and the necessary tools required for dissection, such as forceps, spatula, and scissors, by wiping them clean with an alcohol pad.</li>
 <li>Sacrifice mouse by CO2 asphyxiation in a small gas chamber, followed by immediate decapitation using sharp scissors. Quickly remove the entire brain. Place the brain in beaker of ice-cold sucrose aCSF for approximately 10 minutes.</li>
 <li>In the meantime, prepare Vibratome for slicing. First, place some crushed ice in the specimen bath. Position the specimen chamber and tighten to hold it firmly in place. Add more ice around the specimen chamber to fill in the gaps, making sure no ice gets into the chamber. Place the cleaned razor blade in the blade holder on the Vibratome and fill the specimen chamber with ice-cold sucrose aCSF.</li>
 <li>To set up a working surface for preparing the brain, pour some cold sucrose aCSF on a piece of paper towel that has been placed on an upturned Petri dish. Using forceps transfer the brain onto the prepared Petri dish. For coronal slices, cut the cerebellum along the medial-lateral axis with a razor blade and discard. This creates a flat base that can be affixed to the Vibratome stage.</li>
 <li>Place a drop of the Loctite adhesive (placed in the ice bucket during Step 1) on the specimen stage. Immediately, affix the flat end of the prepared brain on the stage, keeping it as upright as possible. Place the stage in the specimen chamber and tighten the screw, ensuring that the brain is completely immersed in the sucrose aCSF in the chamber.</li>
 <li>Using the controls on the front of the Vibratome adjust the stage so that the razor blade lines up with the top of the brain. Optimal parameters for the Vibratome are obtained by setting the frequency and speed to low. Lower speeds are preferred to minimize the force of the blade from squashing the brain, which is observed at higher speeds. Slice thickness is set to 400 μm.</li>
 <li>The first few slices will not contain the striatum. Repeat the slicing until slices containing the striatum are obtained. Once the striatal region is reached (affirmed by anatomical landmarks), use a paint brush to lift the slice and place in a beaker with oxygenated, room-temperature aCSF. Typically, one can obtain 3 to 4 slices containing the striatal complex so that the caudate-putamen and nucleus accumbens are included.</li>
 <li>Allow the slices to acclimate in oxygenated aCSF at room temperature for at least 1 hour before using for experiments.</li>
</ol>
3. Voltammetric recordings from slices
While the slices are incubating, the slice recording chamber can be prepared.
<ol>
 <li>Take tubing connected to the submersion recording chamber (Custom Scientific, Denver, CO) and place in oxygenating, room temperature aCSF. Set the perfusion pump (Watson Marlow Limited, Falmouth, England) to a flow rate of 1 mL/min. Set the temperature controller to 32 °C. After aCSF fills the custom-built slice holder (modified mesh disc stage), prepare it for the slice by removing any air bubbles using a needle syringe (BD spinal needle).</li>
 <li>Prime the slice bath by drawing a vacuum on the outflow tubing (leading to glass liquid waste bottle) using a syringe to start flow. Place a kimwipe to act as a wick on the edge of the slice holder to control buffer overflow.</li>
 <li>After 1 hour incubation, transfer the slices to the slice holder in the recording chamber, which is continuously perfused with 95% O2/5% CO2 room temperature aCSF.</li>
 <li>Submerge the Ag/AgCl reference electrode in the slice holder (taped to lid of slice chamber) and connect the electrode using an alligator clip to the head stage.
 <ul type="circle">
 <li>The Ag/AgCl reference electrode can be made in house by anodizing (+1 V) a 250 μm silver wire (A-M Systems, Carlsborg, WA) in 1 M HCl for 5 minutes to deposit a thin layer of AgCl on the surface of the silver wire.</li>
 </ul>
 </li>
 <li>Lower the tungsten stimulating electrode (Plastics One, Roanoke, VA) to the surface of the striatal brain slice. The stimulating electrode makes contact with the slice as it rests on top of it, but should not puncture the slice. In our experimental set-up, the stimulation is generated by a Neurolog stimulator.</li>
 <li>The carbon fiber working microelectrode is back-filled with a 150 mM KCl solution using a BD spinal needle. A lead wire is then inserted (Squires Electronics, Cornelius, OR), which is connected to the head stage of the low-noise ChemClamp potentiostat (Dagan Corporations, Minneapolis, MN) using an alligator clip. The working electrode is placed approximately 100 - 200 μm away from the bipolar stimulating electrodes, about 75 μm deep into the slice.</li>
 <li>Electrical stimulations, using either single (monophasic, 350 μA, 60 Hz, and 4 ms pulse width) or multiple (e.g. 5 pulses, 350 μA, 20 Hz, and 4 ms wide) pulses, are delivered by the stimulating electrode to evoke neurotransmitter release 3.</li>
 <li>To measure electrically evoked dopamine using FSCV, a triangle waveform is applied to the electrode. Typical parameters for dopamine detection: potential of the carbon fiber microelectrode is held at -0.4 V versus a Ag/AgCl reference electrode, ramped to a positive limit of +1.2 V, then brought back down to -0.4 V at a scan rate of 400 V/s.</li>
 <li>The slice is electrically stimulated every 5 minutes and voltammetric measurements of the resulting dopamine efflux are made for 15 seconds.</li>
 <li>After at least three stable electrically stimulated dopamine release recordings (difference between peak height is &lt; 10%), aCSF containing pharmacological agent of interest is perfused at a flow rate of 1 mL/min over the slice for 30 minutes to obtain maximal effect. Electrically stimulated dopamine recordings are made every 5 minutes during the pharmacological perfusion.</li>
</ol>
4. Data analysis
Resulting current versus time traces obtained from the slice can be fit by nonlinear regression to a set of Michaelis-Menten based equations, as described by Wightman and colleagues in software written in LabVIEW (National Instruments, Austin, TX) 4-6. In this software, current versus time traces can be fitted by varying two parameters, Vmax (nM/s; corresponding to the rate of uptake by the dopamine transporter as demonstrated by the descending phase of the trace) and dopamine concentration per pulse (nM; corresponding to the peak height maximum). The Km value, reflective of the affinity of dopamine for the dopamine transporter, is set to 160 nM and not changed. The electrode's post-calibration factor that is determined after the experiment is required prior to fitting. The LabView software contains the coefficient of determination (R2) parameter to determine the goodness of fit (R2 values &gt; 0.8 are used).
5. Representative Results
FSCV was used to examine single-pulse, electrically stimulated dopamine release and uptake in the caudate-putamen (CPu), nucleus accumbens (NAc) core, and NAc shell in mice. Representative results shown in Figure 1A demonstrate current (or concentration) versus time plots. The red arrow indicates when electrical stimulation is applied to the slice followed by a corresponding rise in the amount of current attributable to changes in dopamine concentration at the carbon fiber microelectrode. The predominant process during electrical stimulation is dopamine release, but other processes such as uptake and diffusion contribute to the overall observed peak height as well. The descending phase of the peak is mainly attributed to reuptake of the neurotransmitter by its transporter since neuronal stimulation has been stopped 7. However, the peak decay is not limited to reuptake, as diffusion and metabolism also contribute to the decrease in current. It has been postulated that since the time scale of electrochemical measurements is a matter of seconds, FSCV is too fast to measure contributions from metabolism 7. In these current versus time traces, the y-axis is converted to concentration (μM) using the post-experiment calibration factor. The inset for Figure 1A is the respective background subtracted cyclic voltammograms for the current versus time traces. Plotting the measured current (y-axis) against the applied potential to the electrode (x-axis), dopamine is chemically identified with an observed oxidation peak at +0.6 V and a corresponding reduction peak of dopamine-ortho-quinone is observed at -0.2 V versus a Ag/AgCl reference electrode. The third representation of the data uses a three-dimensional pseudo-color plot (Figure 1B) combining the information from both the current versus time traces and the cyclic voltammogram to form a single plot. In the representative pseudo-color plot, time is plotted in seconds along the x-axis, the applied voltage to the carbon fiber working microelectrode is graphed along the y-axis, and current is represented as false color along the z-axis. Due to the small size of carbon fiber microelectrodes (~ 7 μm in diameter), electrically stimulated dopamine dynamics can be detected in discrete anatomical regions of the striatum (CPu versus NAc core versus NAc shell; Figure 2).
An advantage of using striatal coronal slices is that it eliminates contributions from the dopamine cell bodies, and allows for an investigation of presynaptic dopamine dynamics. Presynaptic control of dopamine release and uptake is not strictly limited to dopamine autoreceptor or transporter functions as others have shown 16, 17. Heteroreceptors of other neurotransmitter systems also modulate dopamine dynamics 18, 19. Representative current versus time traces shown in Figure 3A demonstrate that when slices are treated with 1 μM quinpirole (D2/D3 receptor agonist) for 30 minutes, a decrease in electrically-evoked dopamine release is observed. On the other hand, when a substrate for the dopamine transporter, such as methamphetamine, is perfused over the slice for 30 minutes, no difference in dopamine release is observed (Figure 3B). The peak decay is shifted to the right, which is typically associated with alterations in dopamine transporter kinetics (Km) 3. Finally, Figure 3C is a representative trace of the current versus time trace once the slice has been bathed in a 100 ng/mL brain-derived neurotrophic factor (BDNF) solution, which has been hypothesized to influence dopamine release dynamics 20, 21. From this representative trace, it can be seen that BDNF has the ability to enhance electrically-evoked dopamine release. Taken together, these pharmacological treatments emphasize the utility of FSCV to probe dopamine dynamics within the striatum.
The primary limitation of using brain slices to investigate presynaptic dopamine dynamics by FSCV is that the neurocircuitry from an intact brain is lost. With slice FSCV it is impossible to study the effects of neurotransmitters from other brain regions, making it difficult to understand contributions of these systems on the functionality of the region being investigated (e.g. the striatum) or to evaluate non-stimulated dopamine levels. However, recent technical advances in FSCV has allowed for dopamine transient measurements (with and without electrical stimulation) in freely moving rats in response to a pharmacological manipulation, self-administration, or novelty 22 - 24. Overall, slice FSCV provides valuable information on presynaptic dopamine dynamics, and coupling slice FSCV results to complementary neurochemical techniques such as microdialysis, electrophysiology, and/or freely moving FSCV offers a more comprehensive view of neurotransmitter functioning in the brain.
<img alt="Caudate-putamen analysis with voltammetry graphs: voltammogram with current vs. potential chart." src="/files/ftp_upload/3464/3464fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. Electrically-evoked dopamine release measured using FSCV following single-pulse stimulation in dorsal CPu slices from C57Bl/6J mice. (A) The concentration versus time trace in which dopamine release was evoked by a single pulse (red arrow). The single asterisk represents the factors that contribute to the rise in concentration, which is predominantly dopamine release, but uptake and diffusion also contribute. The double asterisk represents the peak signal returning to baseline, mainly due to uptake but also diffusion contributes. Inset displays the corresponding cyclic voltammograms. (B) Representative color plots from the dorsal CPu display time (x-axis), applied potential to the carbon fiber microelectrode versus Ag/AgCl reference electrode (y-axis), and current in pseudo-color.
<img alt="Electrochemical analysis; voltammetry graphs; NAc core/shell; potential vs. Ag/AgCl; current data." src="/files/ftp_upload/3464/3464fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. Dopamine release evoked by a single electrical stimulation pulse (indicated by the red arrow) in the NAc core and shell from C57Bl/6J mice. (A and C) The concentration versus time traces and their corresponding cyclic voltammograms (inset) from the NAc core and shell. (B and D) As previously described, representative color plots from the NAc core and shell.
<img alt="Neurotransmitter release graph; Quinpirole, Methamphetamine, BDNF effects; Baseline comparison." src="/files/ftp_upload/3464/3464fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3. Representative traces after the slice was treated with a pharmacological agent for 30 minutes; in all cases, a single pulse was used to evoke dopamine release in the CPu. (A) Application of the dopamine D2 receptor agonist, quinpirole (red trace) compared to pre-treatment (black trace). (B) Methamphetamine perfusion (purple trace) compared to pre-treatment (black trace). (C) The ability of brain-derived neurotrophic factor to influence dopamine dynamics (blue trace) compared to pre-treatment (black trace).

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<li>Phillips, P. E., Stuber, G. D., Heien, M. L., Wightman, R. M., Carelli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subsecond+dopamine+release+promotes+cocaine+seeking%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Subsecond dopamine release promotes cocaine seeking.</a> Nature. 422, 614-618 (2003).</li>
<li>Robinson, D. L., Heien, M. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Frequency+of+dopamine+concentration+transients+increases+in+dorsal+and+ventral+striatum+of+male+rats+duing+introduction+of+conspecifics%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Frequency of dopamine concentration transients increases in dorsal and ventral striatum of male rats duing introduction of conspecifics.</a> J. Neurosci. 22, 10477-10486 (2002).</li>
</ol>

No conflicts of interest declared.

The protocol presented here demonstrates how to prepare and use mouse coronal brain slices for FSCV experiments. Although this method is specific to obtaining and measuring dopamine dynamics, other neurotransmitters such as adenosine, hydrogen peroxide, norepinephrine, and serotonin have been monitored in vivo or in vitro with FSCV 3, 8 - 11. FSCV can be used to monitor some of these other neurochemicals by simple modifications of the waveform applied to the working electrode 3, 11...

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presynaptic dopamine dynamics in striatal brain slices with fast-scan cyclic voltammetry

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

The overall goal of this procedure is to illustrate a general protocol for slice fast scan cyclic V Telemetry. This is accomplished by first preparing the brain tissue for coronal slicing. Then electrodes are placed onto the discrete brain region of interest.The next step is to deliver voltage as a triangular waveform through the electrode to detect dopamine. Finally, a pharmacological agent of interest is applied to the brain slice and its effect on presynaptic dopamine dynamics is observed. Ultimately, this protocol shows changes in presynaptic dopamine dynamics, such as stimulated dopamine release and dopamine uptake rates through fast scan cyclic vol Telemetry.Though this method can provide insight into presynaptic dopamine dynamics, it can also be applied to other systems, including animal and neurotransmitters, such as rats and fruit flies, and serotonin and norepinephrine respectably demonstrating the procedure will be Francis Mena, MEA Khalid, and Erin oo, the graduate students from my laboratory To begin this procedure, according to the accompanied manuscript, prepare three types of artificial cerebral spinal fluid solutions, sucrose, A-C-S-F-A-C-S-F solution for Volta metric recordings and modified A CSF solution for electrode calibration. Next, transfer 10 milliliters of sucrose A CSF into a small beaker and place it in an ice bucket. Then place the instant Loctite adhesive in an ice bucket.After that, prepare a razor blade and the necessary tools required for dissection, such as forceps, spatula, and scissors by wiping them with an alcohol pad. Then sacrifice a mouse by decapitation and quickly remove its brain. Place the brain in a beaker of ice cold sucrose, A CSF for approximately 10 minutes.In the meantime, prepare the VIBRAM for slicing. Place some crushed ice in the specimen bath. Then place the specimen chamber in the Vibram and fix it firmly.Add more ice around the specimen chamber to fill the gaps and make sure that no ice gets into the chamber. Afterward, place a cleaned razor blade into the blade holder. Then fill the specimen chamber with ice cold sucrose A CSF.Subsequently, pour some cold sucrose A CSF on a piece of paper towel on an upturn Petri dish. Transfer the freshly dissected mouse brain onto it with a pair of forceps. Then remove the cerebellum by cutting along the medial lateral axis with a razor blade.This creates a flat base that can be affixed to the vibram stage. Next, add a drop of the Loctite adhesive on the specimen stage. Affix the flat end of the prepared brain on the stage immediately, and keep it as upright as possible.Then place the stage in the specimen chamber and tighten the screw. Ensure that the brain is completely immersed in the sucrose A CSF in the chamber. After that, adjust the stage with the control so that the razor blade lines up with the top of the brain.Optimal parameters for the vibrator are obtained by setting the frequency and speed to low. Next, set the slice thickness to 400 micrometers. The first few slices usually do not contain the striatum.Repeat the slicing until the slices containing the striatum are obtained. Once the stri AAL region is reached, use a paintbrush to lift the slice and place it in a beaker with oxygenated room temperature. A CSF.Typically, one can obtain three to four slices containing thesal complex in which the caudate putamen and the nucleus accumbens are included. Allow the slices to acclimate in A CSF bubbling with 95%O2 5%CO2 at room temperature for at least one hour before proceeding with the experiment. To begin this procedure, connect the tubing to the submersion recording chamber.Then pump the oxygenated room temperature A CSF at the rate of one milliliter per minute. Set the temperature controller to 32 degrees Celsius after A CSF has filled the custom-built slice holder. Remove any air bubbles under the mesh using a needle syringe.Create negative pressure in the outflow tubing with a syringe in order to start the flow To control the buffer overflow. Place a Kim wipe on the edge of the slice holder to act as a wick. Then transfer a slice to the slice holder in the recording chamber, which is continuously perfused with oxygenated A CSF at room temperature.Submerge the silver silver chloride reference electrode in the slice holder and connect it to the head stage with an alligator clip. Attach the carbon fiber micro electrode to the micro manipulator and connect it to the head stage with an alligator clip. Next, lower the tungsten stimulating electrode and the carbon fiber micro electrode to the surface of the sal brain slice.The stimulating electrode will be used to deliver either a single pulse or multiple pulses of electric stimulations to the slice to evoke neurotransmitter release to measure the electrically evoked dopamine using fast scan cyclic vol telemetry or FSCV, apply voltage in a triangular waveform to the electrode. Hold the potential of the carbon fiber micro electrode at negative 0.4 volts. Ramp it to a positive limit of 1.2 volts.Then bring it back down to negative 0.4 volts at a scan rate of 400 volts per second. Stimulate the slice every five minutes and make the vol metric measurement of the resulting dopamine FLX for 15 seconds each time after at least three. Stable electrically stimulated dopamine release recordings defined as less than 10%difference between peak heights.Switch to perfusing the slice with a CSF containing pharmacological agent of interest at a flow rate of one milliliter per minute for 30 minutes to obtain maximal effect similar to baseline recording record. The electrically stimulated dopamine release every five minutes during the pharmacological perfusion. Figure A is an example of the electrically evoked dopamine release measurement using FSCV, following single pulse stimulation in the caudate putamen.Figure B shows a three dimensional pseudo color plot that combines the information from both the current versus time traces and the cyclic volt Graham. Similarly, figure A and C show the dopamine release evoked by a single electrical stimulation pulse in the nucleus accumbens core and shell respectively, and the three dimensional pseudo color plots for the nucleus accumbens core and shell are indicated in figure B and D respectively. Shown here are the representative traces after the slice had been treated with a pharmacological agent for 30 minutes, followed by single pulse stimulations to evoke dopamine release in the caudate putamen.The agents used in the experiments are quinol, methamphetamine, and brain derived neurotrophic factor. After watching this video, you should have a good understanding of how to prepare mouse coronal brain slices for obtaining and measuring presynaptic dopamine dynamics using fast and likely will telemetry.

presynaptic-dopamine-dynamics-striatal-brain-slices-with-fast-scan

Research

JoVE Journal

Neuroscience

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Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

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Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation

Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex.  Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV).  Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability.  Voltage biasing, EIS, CV, and neural recordings are complementary.

Voltage Biasing, Cyclic Voltammetry, &amp; Electrical Impedance Spectroscopy for Neural Interfaces

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Learn fast-scan cyclic voltammetry for measuring presynaptic dopamine dynamics in striatal brain slices. This method enables real-time observation of dopamine release and uptake in neuroscience research.

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

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