Name: Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration (Video) | JoVE Journal
Uploaded: 2013-07-31T17:00:00.000+00:00
Duration: 7 min 8 s
Description: Learn fast micro-iontophoresis of glutamate and GABA to investigate synaptic integration in neurons. This electrophysiology technique enables precise study of postsynaptic signal processing with high temporal resolution.

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5 minutes

We thank Hans Reiner Polder, Martin Fuhrmann and Walker Jackson for carefully reading the manuscript. The authors received funding that was provided by the ministry of research MIWF of the state Northrhine-Westfalia (S.R.), the BMBF-Projektr&auml;ger DLR US-German collaboration in computational neuroscience (CRCNS; S.R.), Centers of Excellence in Neurodegenerative Diseases (COEN; S.R.), and the University of Bonn intramural funding program (BONFOR; S.R.).

1. System Requirements
<ol>
	<li>Microscope system: Good visualization of the dendrite is crucial. If available, use a two-photon laser scanning microscope system. In our experiments we used a TRIM Scope II, LaVision Biotec, Bielefeld, Germany or an Ultima IV system, Prairie Technologies, Middleton, Wisconsin equipped with a Ti:Sapphire ultrafast-pulsed laser (Chameleon Ultra II, Coherent) and a high NA objective (60X, 0.9 NA, Olympus) to visualize the dendrites which we had filled with a fluorescent dye via the patch pipette. Although photo-damage is thought to be less severe using 2-photon scanning, reduce the laser power (below 8 mW at the tissue) and dwell times (below 1 &mu;sec) or as much as possible.</li>
	<li>A second possibility is to trigger a wide-field fluorescence light source (LED or a fluorescent lamp) synchronously with acquisition to reduce exposure time as much as possible. We have used a monochromator with an integrated light source (TILLPhotonics, Gr&auml;felfing, Germany) on a Zeiss Axioskop 2 FS upright microscope which was equipped with Dodt-contrast infrared illumination (TILLPhotonics, Gr&auml;felfing, Germany). In our experiments exposure times ranged from usually 10 msec to max. 30 msec.</li>
	<li>Use a fast micro-iontophoresis amplifier, e.g. a two-channel micro-iontophoresis system MVCS-C-02 (npi electronic, Tamm, Germany) with fast capacitance compensation. The fine-tipped iontophoresis microelectrodes have resistances of 25 - 100 M&Omega; (crucially depending on the tip size and the pipette shape), and fast rise times can only be achieved if the capacitance compensation of the iontophoretic amplifier is optimally tuned. This fast compensation is necessary to apply current pulses with a brief and rapid onset in the sub-millisecond range to the iontophoretic pipette and thereby to eject the transmitter with high spatial resolution in spot sizes below 1 &mu;m13. Iontophoresis amplifiers are also available from several other companies, which we have not tested in our laboratory. These devices are to our knowledge not equipped with capacitance compensation circuitry.</li>
</ol>
2. Prepare Solutions
<ol>
	<li>Prepare artificial cerebrospinal fluid (ACSF) and internal solution as it is required for the experimental design. The only addition to the internal solution, which is required, is a red or green fluorescent dye (e.g. 50 to 200 &mu;M Alexa Fluor 488 or 594 hydrazide, Invitrogen), depending on the optical equipment. Here an example for ACSFsucrose which can be used for dissection, in mM: 60 NaCl, 100 sucrose, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 CaCl2, 5 MgCl2, 20 glucose; and for normal ACSF solution for patch-clamp experiments in mM: 125 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2.6 CaCl2, 1.3 MgCl2, 15 glucose.</li>
	<li>Carbogenize (95% O2, 5% CO2) all extracellular solutions constantly.</li>
	<li>Prepare internal solution, for example, in mM: 140 K-gluconate, 7 KCl, 5 HEPES-acid, 0.5 MgCl2, 5 Phosphocreatine, 0.16 EGTA; with 50 - 200 &mu;M Alexa 488.</li>
	<li>For glutamate micro-iontophoresis prepare a solution with 150 mM glutamic acid and adjust the pH to 7.0 with NaOH. Add 50 -200 &mu;M Alexa Fluor 488 or 594 hydrazide (Invitrogen) for visualization.</li>
	<li>For iontophoresis of GABA prepare a 1 M GABA solution and adjust the pH to 5 with HCl15. At this pH GABA is charged, only then it can be applied using iontophoresis. Please note that the low pH in the solution ejected to the extracellular space might effect GABA transmission itself 16,17. 
	Protect the GABA solution from light and frequently prepare fresh GABA stock solution, since older solution can lose its effectiveness.</li>
	<li>If it is difficult to see GABAergic events, a high Cl- driving force internal solution, for example by leaving out KCl, might help to visualize the GABAergic events to see if the GABA iontophoresis is working; however to investigate synaptic integration a physiological driving force might be recommended. For general detection of small GABAergic events, a protocol shown in Figure 8C can help.</li>
</ol>
3. Pull and Test the Iontophoresis Pipettes
<ol>
	<li>In general, pulling the right pipettes is maybe the most critical step to achieve controlled neurotransmitter iontophoresis. When using iontophoresis in cell culture, it is possible to pull very fine electrodes similar to sharp microelectrodes10. In patch-clamp experiments in acute slices, however, these very thin pipettes bend on the slice surface when they are lowered into the tissue with an angle, making it impossible to reach deeper dendrites.</li>
	<li>Therefore, pull a pipette with a very small tip, so that no neurotransmitter can leak out, but the tip has to be still rigid enough to penetrate into the tissue (Figure 2). For example, use 150 GB F 8P class pipettes (Science Products, Hofheim, Germany) and a horizontal puller (for example a DMZ-Universal Puller, Zeitz-Instruments GmbH, Martinsried, Germany; or a P-97 Puller, Sutter Instrument Company, Novato, CA) with several pulling steps to achieve a small opening, but also a short tapper with a steep angle (Figure 2 and Table 2).</li>
	<li>It is also possible to use quartz glass pipettes to pull iontophoretic pipettes10. These are supposed to have better mechanical properties and to be more reliable; however special laser pullers are required. But it is also possible to achieve good results with normal glass pipettes, which are usually used for pulling patch pipettes.</li>
	<li>Test the pipette performance and resistance in a chamber without tissue, before using them for the first time, since leakage of glutamate could harm the tissue.</li>
	<li>Set up the iontophoresis amplifier correctly. Then fill the pipette with the neurotransmitter and dye containing solution and place it into the bath (ACSF).</li>
	<li>Compensate the capacitance (Figure 3). Usually very sharp pipettes will have a higher capacitance than blunt ones.</li>
	<li>Check the resistance of the pipette: The micro-iontophoresis amplifier used here has a build-in feature for measuring the pipette resistance. It evokes brief rectangular test pulses, which can be monitored with a standard oscilloscope or an A/D board connected to a computer with acquisition software. Depending on the shape and the tip size, the tip should have a resistance between 25 - 90 M&Omega;.</li>
	<li>Focus on the tip with a 60X or 40X water immersion objective and switch to fluorescent imaging and if possible, zoom in. If fluorescent dye leaks out of the pipette tip, apply a small positive (in the case of glutamate) or negative (in the case of GABA, Figure 4) retain current (&lt;0.02 &mu;A). If that doesn't help to cancel the leakage, change the pipette.</li>
	<li>Apply a strong step current or use the manual trigger and monitor the tip in the fluorescent image to see if the solution can be ejected out of the pipette. As mentioned above, the polarity of the current pulse is depending on the charge of the molecule that is supposed to be ejected. To eject glutamate it is a negative current and for GABA a positive current (Figure 4).</li>
	<li>Air bubbles in the pipette that block ejection of dye and transmitter, can be cleared by applying a high ejection current several times.</li>
	<li>Taken together, if there is no visible leakage and test ejection was successful, compensate the capacitance and start the experiment.</li>
	<li>Caution: Since capacitance compensation is achieved by a feedback circuit, this circuit can overshoot or oscillate if it is overcompensated. Gently use the dial setting for capacitance compensation.</li>
	<li>Depending on the puller stability it is sometimes necessary to adjust the puller settings from time to time, since the filament may have changed properties. However, if once a good pipette is designed, it can be used for several days. Therefore, after finishing the experiment store the iontophoretic pipette in a closed container without damaging the tip.</li>
	<li>For the next experiment fill the pipette with the neurotransmitter solution, apply several strong eject pulses to clear the tip and then check if the properties (e.g. resistance) change significantly. Then compensate the capacitance and use the pipette again. Do not use a single pipette with different neurotransmitter solutions.</li>
</ol>
4. Prepare the Brain Slices
<ol>
	<li>If micro-iontophoresis is used for the first time definitely prepare the slices after establishing a reliably working puller program.</li>
	<li>Perform anaesthesia and decapitation procedures in accordance to the animal care guidelines of your institution or local authority.</li>
	<li>After the removal of the brain, transfer it to ice cold ACSFsucrose (see Protocol 2.1).</li>
	<li>Cut the region of interest into slices of appropriate thickness (for example 300 &mu;m).</li>
	<li>Incubate the slices in ACSFsucrose at 35 &deg;C for 30 min. Subsequently, transfer them to a submerged holding chamber containing normal ACSF at room temperature.</li>
	<li>Throughout the preparation and experiment carbogenize the ACSF surrounding the slices with 95% O2, 5% CO2 .</li>
</ol>
5. Establish a Whole-cell Recording
<ol>
	<li>Position the iontophoresis pipette(s) already near the slice surface before patching a cell, to avoid long lasting positioning, after establishing the whole-cell mode.</li>
	<li>Pull a low resistance patch pipette (3 - 5 m&Omega;), fill it with the dye containing internal solution and apply positive pressure to the pipette (30 - 60 mbar).</li>
	<li>Enter the bath and approach the cell under visual guidance (infrared dodt contrast or two photon gradient contrast image).</li>
	<li>Monitor the pipette resistance with a test pulse (e.g. -10 mV, 20 msec) in voltage clamp mode. When touching the tissue correct the offset potential.</li>
	<li>Approach the cell and gently push the pipette tip into it until a "dimple" can be seen clearly. Immediately release the pressure of the pipette, apply 40 - 60 mbar negative pressure and switch the membrane potential to -65 mV.</li>
	<li>When the holding current reaches values below 100 pA, release the negative pressure.</li>
	<li>After establishing a giga seal (resistance &gt;1 G&Omega;), rupture the membrane with a short, strong suction to the pipette or brief overcompensation of the capacitance compensation circuit.</li>
	<li>Depending on which mode (voltage or current clamp) is required for the experimental design, compensate appropriately.</li>
	<li>Start to bring the iontophoretic pipette into its final position. If a more detailed description of how to successfully perform patch clamp recordings is needed, there are several excellent guidelines available18,19.</li>
</ol>
6. Place the Iontophoretic Pipette and Generate a Postsynaptic Iontophoretic Potential
<ol>
	<li>In general, iontophoretic events can be evoked at defined locations depending on the desired experiment, for example, at a spiny dendrite for glutamate micro-iontophoresis, at the dendritic shaft, soma or axon initial segment for GABA micro-iontophoresis.</li>
	<li>Approach the cell up to approximately 1 &mu;m distance without touching it. After reaching the position of interest it is crucial that no neurotransmitter is leaking out and that the pipette capacitance is compensated correctly.</li>
	<li>If approaching the cell with a glutamate filled iontophoretic pipette causes detectable depolarization of the membrane potential, adjust the retain current if possible, or change the pipette.</li>
	<li>Apply short negative current pulses, starting from zero and increase the current systematically (e.g. 0.1 - 0.4 msec, 0.01 - 1 &mu;A pulses). This helps to find out in which range the iontophoretic current evokes the desired responses in the specific experimental set-up.</li>
	<li>If there is no response detectable, lift the pipette several hundred micrometers and apply a strong eject current (&gt;0.1 &mu;A) to clean the tip. Adjust the capacitance compensation, approach the cell, and try again.</li>
	<li>If there is still no response, reduce the retain current. Be very careful with the dial settings since this procedure can cause uncontrolled neurotransmitter release. Therefore, constantly monitor the recording to detect respective changes in membrane potential.</li>
	<li>If it is difficult to detect GABAergic events, it can be helpful to use an internal solution composition yielding in a high Cl- driving force. To achieve this, reduce the Cl- concentration in the pipette solution (see protocol section 2.6.). This will result in larger GABAergic events at resting membrane potential due to a higher driving force.</li>
	<li>Alternatively, inject long current steps resulting in membrane potential chances from -100 mV to -48 mV (Figure 8C). With this protocol the Cl- driving force is increased at hyperpolarized potentials causing a depolarizing GABA response.</li>
	<li>It is also possible to use the step current injection protocol to determine the reversal potential of the evoked events, which helps to determine the GABAergic nature of the events. Leakage of GABA is harder to detect than leakage of glutamate. In this case, a constant monitoring of the input resistance can help, when the GABA filled pipette is approached. If the input resistance decreases, the retain current can be increased or a different pipette should be used.</li>
	<li>As control experiments for glutamate iontophoresis, we suggest a Ca2+ imaging experiment, using 200 &mu;M OGB-1 and no EGTA in the pipette solution to visualize local calcium influx that might be caused by leaking glutamate.</li>
	<li>In general, to achieve a stable response it is very important to have a mechanically stable pipette to avoid drift over time. Drift can be caused by temperature changes, therefore it is recommended to switch on the equipment at least half an hour before the measurements to avoid thermal drift. Be sure to use good cartridge seals in the pipette holder and the tip is really fixed. The holder itself can be additionally fixed with Teflon tape; furthermore, be sure that there is no tension on the cables from the headstage or manipulator, which also is a potential source of drift.</li>
</ol>

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The authors declare that they have no competing financial interests.

Here we explain how to apply fast micro-iontophoresis of neurotransmitters to investigate synaptic integration on dendrites. This technique has been successfully used to investigate glutamatergic and GABAergic synaptic transmission in different brain regions in vitro and in vivo9,20-22. Micro-iontophoresis has been used for more than 60 years, but in early years it was mostly used to either locally apply neurotransmitters and drugs on slow or intermediate timescales23 or for microi...

Neurons in the central nervous system receive a variety of synaptic inputs on their thin and ramified dendritic processes1. There, the majority of the excitatory dendritic inputs are mediated by glutamatergic synapses. These synapses can be activated in a spatially distributed way, resulting in postsynaptic linear integration of excitatory postsynaptic potentials (EPSPs). If the synapses are activated simultaneously and in spatial proximity on the dendrite, these excitatory inputs can be integrated supra-linearly and generate dendritic spikes2-5.
Furthermore, the integration of excitatory inputs depends on the location of the input on the dendritic tree. Signals that arrive at the distal tuft region are much more attenuated than proximal inputs due to cable filtering6. In the hippocampus, distant inputs to the apical tuft dendrites are generated by a different brain region than those on proximal dendrites7. An exciting question is therefore, how synaptic input is processed by different dendritic compartments and if dendritic integration regulates the influence of these layered inputs on neuronal firing in different ways.
Not only the functional properties, morphological features of the dendrite, the location and clustering of the inputs are affecting the dendritic integration of excitatory inputs, also the additional inhibitory inputs from GABAergic terminals crucially determine the efficacy of the glutamatergic synapses8,9. These different aspects of synaptic integration can be ideally investigated using neurotransmitter micro-iontophoresis, which allows spatially defined application of different neurotransmitters to dendritic domains. We demonstrate here how to successfully establish micro-iontophoresis of glutamate and GABA to investigate signal integration in neurons.
For this application, fine-tipped high resistance pipettes filled with concentrated neurotransmitter solutions are used. These pipettes are positioned close to the outer membrane of the cell, where the neurotransmitter receptors are located. A good visualization of the dendritic branches is required. This is best achieved using fluorescent dyes, which are introduced via the patch pipette. Then a very short (&lt;1 msec) current pulse (in the range of 10 - 100 nA) is used to eject the charged neurotransmitter molecules. With these short pulses and effective capacitance compensation, postsynaptic potentials or currents can be evoked with high temporal and spatial precision, which means the location of the excitatory input is precisely known. Glutamate micro-iontophoresis can activate synapses in a defined radius, which is smaller than 6 &mu;m as shown here (Figure 19), but it is also possible to reach single synapse resolution10-12.
Heine, M., et al showed that the spatial resolution of fast micro-iontophoresis can even be adjusted to spot sizes below 0.5 &mu;m, which is smaller than spot sizes regularly achieved with two-photon uncaging of glutamate13. With fast micro-iontophoresis it is easily possible to use two or more iontophoretic pipettes and place them at different, even distant spots on the dendritic tree. In this way, integration of excitatory events, including those from different pathways, can be investigated. It is also possible to use a glutamate and a GABA filled iontophoretic pipette at the same time. In this way the effect of GABAergic inhibition at different locations relative to the excitatory input (on-path, off-path inhibition) can be studied. Also, the impact of inhibition by interneurons targeting specific neuronal domains, like distal dendrites, soma or axons14, can be investigated using GABA iontophoresis. In cultured neurons, fast micro-iontophoresis offers the opportunity to investigate single synapse distribution and the elementary aspects of synaptic communication in neurons in much more detail10,11.
In this article we demonstrate in detail how to establish glutamate and GABA iontophoresis for the use in acute brain slices, which allows investigating synaptic integration of excitatory and inhibitory inputs in dependence of input location, input strengths, and timing, alone or in interplay. We will point out the advantages and limitations of this technique and how to troubleshoot successfully.

<table><tbody><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>Two-photon laser scanning microscope (TRIM Scope II), and Ultima IV, Prairie Technologies, Middleton, Wisconsin)</td><td>LaVision Biotec, Bielefeld, Germany</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Two-photon laser scanning microscope Ultima IV</td><td>Prairie Technologies, Middleton, Wisconsin, USA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Ti:Sapphire ultrafast-pulsed laser</td><td>Chameleon Ultra II, Coherent</td><td></td><td>&amp;nbsp;</td></tr><tr><td>60X Objective, NA 0.9</td><td>Olympus</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Zeiss Axioskop 2 FS upright microscope</td><td>TILLPhotonics, Gr&amp;auml;felfing, Germany</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Monochromator</td><td>TILLPhotonics, Gr&amp;auml;felfing, Germany</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Micro-iontophoresis system MVCS-02</td><td>NPI Electronics, Tamm, Germany</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Sutter puller P-97</td><td>Sutter Instrument Company, Novato, CA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Glass filaments (150 GB F 8P)</td><td>Science Products, Hofheim, Germany</td><td></td><td>&amp;nbsp;</td></tr><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Alexa Fluor 488 hydrazide</td><td>Molecular Probes life technologies</td><td>A-10436</td><td>&amp;nbsp;</td></tr><tr><td>Alexa Fluor 594</td><td>Molecular Probes life technologies</td><td>A-10438</td><td>&amp;nbsp;</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653</td><td>&amp;nbsp;</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333</td><td>&amp;nbsp;</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S8282</td><td>&amp;nbsp;</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S6297</td><td>&amp;nbsp;</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td>&amp;nbsp;</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C5080</td><td>&amp;nbsp;</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M2670</td><td>&amp;nbsp;</td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>G7528</td><td>&amp;nbsp;</td></tr><tr><td>K-Gluconate</td><td>Sigma Aldrich</td><td>G4500</td><td>&amp;nbsp;</td></tr><tr><td>HEPES-acid</td><td>Sigma Aldrich</td><td>H4034</td><td>&amp;nbsp;</td></tr><tr><td>Phosphocreatin</td><td>Sigma Aldrich</td><td>P7936</td><td>&amp;nbsp;</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E3889</td><td>&amp;nbsp;</td></tr><tr><td>Glutamic acid</td><td>Sigma Aldrich</td><td>G8415</td><td>&amp;nbsp;</td></tr><tr><td>GABA</td><td>Sigma Aldrich</td><td>A5835</td><td>&amp;nbsp;</td></tr><tr><td>NaOH</td><td>Merck</td><td>1.09137.1000</td><td>&amp;nbsp;</td></tr><tr><td>HCl</td><td>Merck</td><td>1.09108.1000</td><td>&amp;nbsp;</td></tr></tbody></table>

fast micro-iontophoresis of glutamate and gaba: a useful tool to investigate synaptic integration

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

A simple approach to determine the spatial spread of iontophoresis is to retract the iontophoretic pipette stepwise from the dendrite, while keeping the ejected glutamate constant. We found that the spatial extent of a micro-iontophoretic stimulation had a diameter of approximately 12 μm (Figure 1 showing radius). How deep in the tissue the iontophoresis can be used depends on the rigidity of the pipette. However, the iontophoretic pipettes needed for experiments in slices (Figure 2), wh...

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The overall goal of this procedure is to understand the integration of inputs along the very complex structure of the neuronal dendritic tree, which finally leads to the neuronal output of action potentials at the axon. This is accomplished by first pulling the appropriate ion aporetic pipettes. The second step is to test the ion aporetic pipettes and to correctly compensate their capacitance.Next, establish the whole cell configuration. The final step is to approach the dendrite and to evoke post-synaptic potentials in the recorded cell. Ultimately, fast micro iontophoresis allows investigating the integration of multiple inputs, which can also belong to different neurotransmitter systems.This method can help to answer key questions in the neuroscientific field, like how inhibitory and excitatory inputs are integrated, and under which conditions. This can lead to neuronal output. Begin this procedure by pulling pipettes with very small tips for controlled neurotransmitter iontophoresis.With a horizontal or vertical pipette puller, small fine tips can be achieved with several pulling steps. However, the tip should still be rigid enough to penetrate into the tissue. Next, set up the ion aporetic amplifier to test the pipette performance in a chamber without tissue.Fill the pipette with neurotransmitter and dye containing solution. Then place it into the A CSF. Subsequently compensate the capacitance with the amplifier.Usually very sharp pipettes will have a higher capacitance than the blunt ones after their capacitance is compensated correctly. Check the resistance of the pipette with the built-in feature of the micro iontophoresis amplifier. The pipette should have a resistance between 25 and 90 mega ohms.Afterward, focus on the pipette tip with a 60 x or 40 x water immersion objective. Then switch to fluorescent imaging. If fluorescent D leaks out of the pipette tip, apply a small positive retain current in the case of glutamate or negative retain current in the case of gaba.If that doesn't help to cancel the leakage, change the pipette. Next, apply a strong step current to the pipette or use the manual trigger Monitor the fluorescent image of the pipette tip to see if the solution can be ejected. If there is no visible leakage and test ejection is successful, check the capacitance compensation and start the experiment.In general, ion aporetic events can be evoked at defined locations depending on the desired experiment. For example, at a spiny dendrite for glutamate micro phoresis, or at the dendritic shaft, soma or axon initial segment for GABA micro phoresis. After establishing the whole cell configuration, place the ion aporetic pipette tip to approximately one micrometer distance from the dendrite or other compartment of interest.Make sure that no neurotransmitter is leaking out and that the pipette and the capacitance are compensated correctly. If approaching the cell with a glutamate filled ion aporetic pipette causes detectable depolarization of the membrane, potential glutamate is leaking out of the pipette. Try to adjust the retain current or change the ion aporetic pipette.Next, apply short negative pulses to the ion aporetic pipette for glutamate current starting from zero and increase the current systematically. This helps to find out the range in which the ion aporetic current evokes the desired responses in the specific experimental setup. If there is no detectable response, lift the ion aporetic pipette several hundred micrometers, and apply a strong current larger than 0.1 microamps to clean the tip.Then adjust the capacitance compensation. Again, approach the cell and try again. If there is still no response, try to reduce the retain current.However, be very careful with the reduction of the retain current since this can cause uncontrolled neurotransmitter release. Therefore, constantly monitor the recording to detect respective changes in membrane potential. If glutamate leaks out of the pipette, reject the pipette for gaba micro iontophoresis.Fill a new pipette with GABA solution. It is very important to have the correct polarity for the ejection current when changing the transmitter in the pipette. For GABA ejection, change the settings to normal resulting in a positive ejection current to detect the GABAergic events more easily.Inject long current steps resulting in membrane potential changes from around negative 100 millivolts to negative 48 millivolts. With this protocol, the chloride ion driving force can be increased, making the events more prominent, and the equilibrium of the event is revealed. This schematic drawing shows single glutamate micro iontophoresis.The EPSP shown here is evoked in a CA one pyramidal neuron with glutamate iontophoresis, and the dendritic sodium calcium spike depicted here is evoked at a dendrite of a CA one pyramidal neuron. The lower trace shows the slope of the voltage trace, and the peak indicates the peak slope of the dendritic spike. When increasing current applied to the ionophore pipette, the EPSP amplitude increases until it crosses the action potential threshold.The schematic drawing here shows double glutamate micro iontophoresis at different locations on the dendritic tree. This ion retic EPSP is evoked at a proximal dendri of a CA one pyramidal neuron, and this one is evoked at a distal dite. This schematic drawing shows GABA micro phoresis.The retic IPSP shown here is evoked at a proximal dendrite of a CA one pyramidal neuron and long current injection with different amplitudes applied to a CA one pyramidal neuron via the patch pipette helps to determine the reversal potential of the evoked event the evoked event reverses at approximately negative 70 millivolts. Lastly, this schematic drawing shows the simultaneous glutamate and GABA iontophoresis that investigates the integration of excitation and inhibition. The dendritic spikes evoked by retic application of glutamate alone.In subsequent sweeps are shown here. The peaks of the lower trace showing the slope of the voltage trace indicate the occurrence of the dendritic spikes, and here is ionically evoked GABAergic IPSP alone. Lastly, both the glutamatergic and GABAergic events together are evoked, which leads to the inhibition of dendritic spikes.After watching this video, it should be very easy to establish fast micro resis in your lab and to gain insight how synaptic inputs are integrated in neurons.

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Research

JoVE Journal

Neuroscience

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Learn fast micro-iontophoresis of glutamate and GABA to investigate synaptic integration in neurons. This electrophysiology technique enables precise study of postsynaptic signal processing with high temporal resolution.

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

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