Name: single cell measurement of dopamine release with simultaneous voltage-clamp and amperometry
Uploaded: 2012-11-21T12:00:00
Description: Watch this Scientific Journal Video about Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry at JoVE.com

We thank Dr. Sanika Chirwa for critical review of this manuscript. This work was supported by National Institutes of Health (DA026947, DA021471, and NS071122).

1. Equipment and Supplies
<ol>
	<li>Mount a Faraday cage on top of the anti vibration table (TMI) to decrease the background noise.</li>
	<li>The simultaneous patch clamp amperometry recording system requires an inverted microscope with excellent DIC optics and a long working distance lens. Connect the microscope lighthouse to a car battery. This DC light source for the system will further decrease the electrical noise.</li>
	<li>Hydraulic micromanipulators (Siskiyou) further decrease noise. In our configuration, we us...

<ol>
	<li>Michael, A. C., Ikeda, M., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+contributing+to+the+recovery+of+striatal+releasable+dopamine+following+MFB+stimulation.">Mechanisms contributing to the recovery of striatal releasable dopamine following MFB stimulation.</a> Brain Res. 421, 325-335 (1987).</li><li>Gonon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+and+extrasynaptic+excitatory+action+of+dopamine+mediated+by+D1+receptors+in+the+rat+striatum+in+vivo.">Prolonged and extrasynaptic excitatory action of dopamine mediated by D1 receptors in the rat striatum in vivo.</a> J. Neurosci. 17, 5972-5978 (1997).</li><li>Sulzer, D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+quantal+size+by+presynaptic+mechanisms.">Regulation of quantal size by presynaptic mechanisms.</a> Rev. Neurosci. 11, 159-212 (2000).</li><li>Napolitano, A., Cesura, A. M., Da Prada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+monoamine+oxidase+and+catechol+O-methyltransferase+in+dopaminergic+neurotransmission.">The role of monoamine oxidase and catechol O-methyltransferase in dopaminergic neurotransmission.</a> J. Neural. Transm. Suppl. 45, 35-45 (1995).</li><li>Sulzer, D., Maidment, N. T., Rayport, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+other+weak+bases+act+to+promote+reverse+transport+of+dopamine+in+ventral+midbrain+neurons.">Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons.</a> J. Neurochem. 60, 527-535 (1993).</li><li>Salahpour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4405-4410 (2008).</li><li>Khoshbouei, H., Wang, H., Lechleiter, J. D., Javitch, J. A., Galli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+dopamine+efflux.+A+voltage-sensitive+and+intracellular+Na+-dependent+mechanism.">Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.</a> J. Biol. Chem. 278, 12070-12077 (2003).</li><li>Goodwin, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+methamphetamine+differentially+affect+dopamine+transporters+in+vitro+and+in.">Amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in.</a> , 284-2978 (2009).</li><li>Kahlig, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+induces+dopamine+efflux+through+a+dopamine+transporter+channel.">Amphetamine induces dopamine efflux through a dopamine transporter channel.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 3495-3500 (2005).</li><li>Swant, J., Chirwa, S., Stanwood, G., Khoshbouei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+reduces+LTP+and+increases+baseline+synaptic+transmission+in+the+CA1+region+of+mouse+hippocampus.">Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus.</a> PLoS One. 5, e11382 (2010).</li><li>Gnegy, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++regulates+amphetamine-induced+dopamine+efflux+and+currents+mediated+by+the+human+dopamine+transporter.">Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter.</a> Mol. Pharmacol. 66, 137-143 (2004).</li></ol>

Simultaneous voltage-clamp and amperometry has the following benefits. All cell types are accessible and can be used for recording. The identification of the cells or neuron where the recordings are done is simple and straightforward. In particular, if the cell is fluorescently labeled by adding a fluorescent tag to the protein of interest the experimenter can easily select the target cell or neuron. The experimental configuration allows uniform and controlled delivery of pharmacological agents either via the patch pipet...

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Anti-vibration table w/faraday cage</td>
			<td>Technical Manufacturing Corporation</td>
			<td>63-500 series</td>
			<td>we use model 63-543</td>
		</tr>
		<tr>
			<td>Inverted microscope Nikon TE-2000</td>
			<td>Nikon</td>
			<td>discontinued</td>
			<td>now Eclipse Ti</td>
		</tr>
		<tr>
			<td>Two low noise amplifiers axopatch 200b</td>
			<td>Molecular Devices</td>
			<td>&nbsp;</td>
			<td>800-635-5577</td>
		</tr>
		<tr>
			<td>1-CV 203 BU headstage</td>
			<td>Molecular Devices</td>
			<td>&nbsp;</td>
			<td>800-635-5578</td>
		</tr>
		<tr>
			<td>1-HL-U pipette holder</td>
			<td>Molecular Devices</td>
			<td>&nbsp;</td>
			<td>800-635-5579</td>
		</tr>
		<tr>
			<td>Digidata 1440A A/D converter</td>
			<td>Molecular Devices</td>
			<td>&nbsp;</td>
			<td>800-635-5580</td>
		</tr>
		<tr>
			<td>Two manipulators Siskyou, left and right handed</td>
			<td>Siskiyou</td>
			<td>MX6600R MX6600L</td>
			<td>877-313-6418</td>
		</tr>
		<tr>
			<td>Laser pipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-2000</td>
			<td>888-883-0128</td>
		</tr>
		<tr>
			<td>Low noise carbon fiber amperometric electrode</td>
			<td>ProCFE</td>
			<td>&nbsp;</td>
			<td><a href="http://www.dagan.com" target="_blank">www.dagan.com</a></td>
		</tr>
		<tr>
			<td>Low noise quartz pipette</td>
			<td>Sutter Instruments</td>
			<td>QF100-70-7.5</td>
			<td>888-883-0128</td>
		</tr>
		<tr>
			<td>12-volt car battery</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>widely available</td>
		</tr>
		<tr>
			<td>Car battery charger</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>widely available</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2&bull;2H20)</td>
			<td>Sigma</td>
			<td>223506</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2&bull;6H20)</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E0396</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

single cell measurement of dopamine release with simultaneous voltage-clamp and amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

Combined patch clamp with amperometry can measure voltage-dependent DAT-mediated DA efflux. Figure 2A shows a representative experimental configuration and recording of DAT-mediated DA efflux when the intracellular milieu and the membrane potential are clamped by a whole-cell patch pipette. Using this technique, cells expressing YFP-DAT proteins are voltage-clamped with a whole cell patch pipette while an amperometric electrode is placed onto the plasma membrane (Figure 2A). The whole ce...

Watch this Scientific Journal Video about Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry at JoVE.com

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

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.

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Research

JoVE Journal

Neuroscience

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

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

Presynaptiske Dopamin Dynamics i striatale banene Brain Slices med Fast-scan Syklisk 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 ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Encellet Profiling for å utvikle og modne Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Optisk registrering av suprathreshold nevrale aktiviteten med encellede og single-spike oppløsning

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Samtidig Electroencephalography, Real-time Måling av laktat Konsentrasjon og Optogenetic Manipulering av neuronal aktivitet i Rodent Cerebral Cortex

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

Dyp hjernestimulering med Simultaneous fMRI i Gnagere

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Utarbeidelse av Neuronal Co-kulturer med Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

Samtidig Påvisning av c-Fos Aktivering fra mesolimbiske og Mesocortical Dopamin Reward nettsteder Etter Naive sukker og fett Svelging i rotter

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Elektrofysiologisk metode for helcelle spenningsklemmeopptak fra<em&gt; Drosophila</em&gt; Fotoreseptorer

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Levator Auris Longus Forberedelse for undersøkelse av pattedyr nevromuskulær overføring Under spenning klemme forhold

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...