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 microinjection of substances into cells24.
Micro-iontophoresis became a particularly interesting tool for studying dendritic integration since the introduction of amplifiers, which are equipped with a fast capacitance compensation for high-resistance electrodes10,11. With this improvement it became possible to apply brief rectangular iontophoretic currents resulting in postsynaptic responses, which resembled more realistically the time course of synaptic events10,25.
What are the technical requirements and difficulties to deal with, when establishing micro-iontophoresis? An iontophoretic amplifier is needed that allows for compensation of the high capacitance of the fine iontophoretic pipettes. The correct tuning of the capacity compensation is very important if high speed operation in conjunction with high resistance microelectrodes is required. Uncompensated stray capacitances are charged from the iontophoretic current that is supplied by the instrument, and therefore slows application. Compensating the capacitance properly is crucial and explained in detail in the video. Additionally, it is critical that the optical system gives a good fluorescent image of the dendrite and the iontophoretic pipette without inducing photo-damage of the tissue. Optimally, use a two-photon system and reduce laser power and dwell times as much as compatible with a decent image quality. If a more conventional light source is used, make sure to reduce the exposure time as much as possible, for example, by using mechanical shutters or electrical triggering. Probably the most critical point is the pipette design, which will allow for a defined and specific application of the neurotransmitter used. This step does require some effort and time to find the optimal settings. Here, we present protocols for glutamate and GABA iontophoresis, if other neurotransmitters, blockers or modulators are used, it is necessary that the substance is highly concentrated and even more importantly that it is charged. For example, since GABA has a net charge of zero at a pH of 0, the pH has to be adjusted (see Protocol section 2), resulting in a net positive charge.
When micro-iontophoresis is established, it will provide an extended toolset to study synaptic integration in neurons. It allows stimulating synapses of interest selectively with a particular neurotransmitter. Using a selected neurotransmitter on defined locations, postsynaptic currents and potentials and their propagation can be investigated. Furthermore, it is possible to use multiple iontophoretic electrodes simultaneously to investigate the integration of several glutamatergic inputs or systematically change the relative timing or the strength of events.
Some general critical points have to be considered when using micro-iontophoresis. The profile of neurotransmitter concentration after micro-iontophoresis is different from endogenous release. Murnick and others10 reported that the speed of release from the iontophoresis tip (about 1 msec) is likely to be significantly slower than synaptic release from vesicles, which occurs in fractions of a millisecond (0.2 msec)26. Also, diffusion to and from the synaptic cleft is likely slowing concentration rise and decay of iontophoretically applied neurotransmitters. Furthermore, the volume of ejected neurotransmitter is likely to be higher than physiologically released volumes, however, with high-resistance electrodes, single glutamatergic and GABAergic postsynapses could be activated using micro-iontophoresis 10-12. A spatial selectivity in the range of a few micrometers has also recently been achieved by two-photon uncaging of neurotransmitters 3,4,27.
While considerably more cost intensive, two-photon uncaging has several advantages over micro-iontophoresis. In particular it can be used to release neurotransmitter simultaneously at multiple synaptic sites and does not require the precise positioning of a fine electrode tip. However, it also has some important disadvantages to be considered when selecting a method for a particular experiment. Many caged compounds have unwanted side effects such as the blockade of neurotransmitter receptors28. For example, many glutamate and GABA cages have been reported to interfere with GABAergic inhibition. Also, the relatively high laser power required for two-photon uncaging may result in photo-damage of the postsynaptic neuron, particularly when the stimulation is applied repeatedly over several minutes. Furthermore, while micro-iontophoresis is limited in the number of stimulated sites, these sites can be chosen freely on the whole dendritic tree of a neuron, for example on distal and basal dendrites, to simulate layered synaptic input from different brain regions. Two-photon uncaging, as it is currently used by most groups, will be limited to synaptic sites in a particular focal plane and in the field of view, which depends on the objective used (typically 40X or 60X water immersion). It has to be considered that both techniques always lead to activation of extrasynaptic receptors, which may make the interpretation of the results more difficult.
Another disadvantage of micro-iontophoresis is that only the postsynapse can be activated. If presynaptic function and the role of vesicle-released neurotransmitter need to be set into the experimental focus, local electrical synaptic stimulation may be a method of choice. However, the disadvantages of electrical synaptic stimulation compared to micro-iontophoresis are the unknown location of the activated synapses and the co-activation of axons from different neuronal populations, potentially belonging to other neurotransmitter systems (Table 1).
In summary the most important advantage of micro-iontophoresis is, in our view, that it is possible to use several different neurotransmitters and to study their interaction on neuronal compartments such as dendrites9.

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

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 &mu;m (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), which are used here, are not limiting the depth of penetration. Rather the optical system and the decreasing resolution in the depth of the brain slice is the limiting factor. For a good quality recording it is crucial that no transmitter is leaking out of the pipette. In the case of glutamate, a leakage can be identified if there is a sudden depolarisation when the dendrite is approached with the pipette tip or if the baseline suddenly becomes unstable (Figure 5). After establishing a stable recording, it is possible to evoke EPSPs of defined amplitudes, dendritic spikes or action potentials with this technique on any location throughout the dendritic tree (Figure 6). By applying glutamate with fast micro-iontophoresis, the properties of EPSPs, their propagation and summation, simultaneously at different even distant locations, can be investigated (Figure 7). GABAergic events can be evoked alone or in addition to glutamate with a second pipette by filling the iontophoretic pipette with a highly concentrated GABA solution (see: Protocol section 2) and a positive eject current. There is a simple protocol to confirm the GABAergic nature of the events and to make GABAergic events easier to detect, if not using a high driving force internal solution: Inject negative currents (approx. 1 sec; the amplitude depends on the input resistance of the cell) resulting in hyperpolarising voltage steps, starting at around -100 mV and then increase in 5 mV steps (Figure 8). At very negative potentials the GABAergic events are easier to detect, due to the higher driving force. And if the signals reverse around the calculated Cl- reversal potential for the solutions, it is very likely that they are GABAergic in nature (Figure 8). With an additional GABA micro-iontophoretic pipette, it is possible to investigate, for example, the effect of GABAergic inhibition on glutamatergic events, like dendritic sodium/calcium spikes, by varying the relative timing of dendritic spike and IPSP (Figure 9), the relative location of both events, or their amplitudes. (All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of the University of Bonn, the German Center for Neurodegenerative Diseases and the state Northrhine-Westfalia.)
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50701/50701fig1highres.jpg" src="/files/ftp_upload/50701/50701fig1.jpg" /> 
 Figure 1. Spatial extent of iontophoretically ejected glutamate determined by pipette retraction. A) Maximum intensity projection of a two-photon image of a CA1 pyramidal cell dendrite filled with 100 &mu;M Alexa 594 and the iontophoretic pipette in the initial position (scale bar 8 &mu;m). Insets show retraction of the iontophoretic pipette and the corresponding iontophoretic EPSP recorded at the soma. Arrow indicates the position of the iontophoretic pipette tip. B) Distance dependence of EPSP amplitudes relative to the initial position of the iontophoretic pipette tip (&le;1 &mu;m away from dendrite, n = 6 branches). With this we could estimate the radius of the glutamate spread by systematically retracting the pipette. Inset shows representative example traces. Error bars represent mean &plusmn; SEM. (Adapted from M&uuml;ller et al. 20129, reprinted with permission from Elsevier).
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50701/50701fig2highres.jpg" src="/files/ftp_upload/50701/50701fig2.jpg" /> 
 Figure 2. How an iontophoretic pipette should look like. A) Infrared CCD image of an iontophoretic pipette compared to a small 5.5 M&Omega; patch pipette using a 60X objective. B) Iontophoretic pipette with scale using a 60X objective. Calibration: smallest = 10 &mu;m.
<img alt="Figure 3" fo:content-width="4in" fo:src="/files/ftp_upload/50701/50701fig3highres.jpg" src="/files/ftp_upload/50701/50701fig3.jpg" /> 
 Figure 3. Capacitance compensation of the iontophoretic pipette using the build in test-pulse (10 nA, 10 msec, npi electronic, Tamm, Germany). It is important to compensate the capacitance correctly to monitor the right pipette resistance and to ensure fast and accurate neurotransmitter application.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/50701/50701fig4highres.jpg" src="/files/ftp_upload/50701/50701fig4.jpg" /> 
 Figure 4. Principles of micro-iontophoresis. A) Schematic drawing of a CA1 pyramidal neuron in whole cell patch-clamp configuration and an iontophoretic pipette filled with glutamate. Glutamate is negatively charged, therefore a positive current applied to the pipette will keep it from leaking out of the pipette: The retain current is positive (left panel). To eject glutamate from the pipette, apply a negative current (right panel). In this way glutamate is forced out of the pipette and can evoke excitatory events in the postsynaptic cell. B) Schematic drawing of CA1 pyramidal neuron in whole cell patch-clamp configuration and an iontophoretic pipette filled with GABA. GABA is positively charged at a low pH. Therefore, a negative current will keep it from leaking out of the pipette (left panel). To eject GABA apply a positive current (right panel). In this way GABA comes out of the pipette and can evoke inhibitory events in the postsynaptic cell.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/50701/50701fig5highres.jpg" src="/files/ftp_upload/50701/50701fig5.jpg" /> 
 Figure 5. Good and bad iontophoretic pipettes. A) Representative example of an iontophoretic glutamatergic EPSP generated on a dendritic branch of a CA1 pyramidal neuron. B) Representative example of an iontophoretic dendritic spike generated a dendritic branch in the CA1 area of the hippocampus with ongoing mild glutamate leakage from the pipette tip.
<img alt="Figure 6" fo:content-width="6in" fo:src="/files/ftp_upload/50701/50701fig6highres.jpg" src="/files/ftp_upload/50701/50701fig6.jpg" /> 
 Figure 6. Representative results for single glutamate micro-iontophoresis. A) Schematic drawing of a patched CA1 pyramidal neuron and an iontophoretic pipette filled with glutamate. B) EPSP evoked in a CA1 pyramidal neuron with glutamate iontophoresis. C) Dendritic Na+/Ca2+spike evoked on a proximal dendrite of a CA1 pyramidal neuron, lower trace shows the slope of the voltage trace, peak indicates the peak slope of the dendritic spike. D) When increasing the current applied to the iontophoretic the amplitude of the EPSP will increase until it crosses the action potential threshold.
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/50701/50701fig7highres.jpg" src="/files/ftp_upload/50701/50701fig7.jpg" /> 
 Figure 7. Representative results for double glutamate micro-iontophoresis at different locations on the dendritic tree. A) Schematic drawing of a patched CA1 pyramidal neuron and two iontophoretic pipettes filled with glutamate, which are positioned on a proximal, and a distal dendrite, respectively. B) Iontophoretic EPSP evoked on a proximal dendrite of a CA1 pyramidal neuron (1). C) Iontophoretic EPSP evoked at a distal dendrite (2).
<img alt="Figure 8" fo:content-width="5in" fo:src="/files/ftp_upload/50701/50701fig8highres.jpg" src="/files/ftp_upload/50701/50701fig8.jpg" /> 
 Figure 8. Representative results for GABA micro-iontophoresis. A) Schematic drawing of a patched CA1 pyramidal neuron and an iontophoretic pipette filled with GABA. B) Iontophoretic IPSP evoked on a proximal dendrite of a CA1 pyramidal neuron. C) Long current injection of systematically changing amplitudes to a CA1 pyramidal neuron via the patch pipette, to determine the reversal potential of the evoked event (current injections from -400 pA to 0 pA). Artifact indicates time-point of GABA iontophoresis. The evoked event indicates a reversal potential of approximately -70 mV (arrow).
<img alt="Figure 9" fo:content-width="6in" fo:src="/files/ftp_upload/50701/50701fig9highres.jpg" src="/files/ftp_upload/50701/50701fig9.jpg" /> 
 Figure 9. Representative results of simultaneous glutamate and GABA iontophoresis to investigate integration of inhibition and excitation. A) Schematic drawing of a patched pyramidal neuron and one pipette filled with glutamate (green) and on with GABA (orange). B) Iontophoretically evoked dendritic spikes alone, in subsequent sweeps, lower traces show dV/dt, peaks indicate dendritic spikes. C) Iontophoretically evoked IPSP alone. D) Both, glutamatergic and GABAergic events together.
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Location specificity</td>
			<td>Transmitter specificity</td>
			<td>Toxicity / side effects</td>
			<td>Presynaptic stimulation</td>
			<td>Long-term experiment</td>
			<td>Costs/complexity</td>
		</tr>
		<tr>
			<td>Micro- iontophoresis</td>
			<td>++</td>
			<td>+++</td>
			<td>++</td>
			<td>-</td>
			<td>++</td>
			<td>++</td>
		</tr>
		<tr>
			<td>2-photon uncaging</td>
			<td>+++</td>
			<td>+++</td>
			<td>+</td>
			<td>-</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>Synaptic stimulation</td>
			<td>-</td>
			<td>-</td>
			<td>+++</td>
			<td>+++</td>
			<td>+++</td>
			<td>+++</td>
		</tr>
	</tbody>
</table>
Table 1. Comparison of different techniques. Advantages and disadvantages of techniques to stimulate neurons according to different criteria (- = bad, + = not optimal, ++ = good, +++ = best).
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td colspan="2">Patch pipettes</td>
			<td colspan="2">Iontophoretic pipettes</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Pre-pulls P (A) Single pull</td>
			<td>Last Pull P(B)</td>
			<td>Pre-pulls P(A) Single Pull</td>
			<td>Last Pull P(B)</td>
		</tr>
		<tr>
			<td>Heat H</td>
			<td>700</td>
			<td>480</td>
			<td>510</td>
			<td>600</td>
		</tr>
		<tr>
			<td>Force Pre Pull F(TH)</td>
			<td>018</td>
			<td>035</td>
			<td>018</td>
			<td>008</td>
		</tr>
		<tr>
			<td>Distance Threshold s(TH)</td>
			<td>017</td>
			<td>012</td>
			<td>025</td>
			<td>015</td>
		</tr>
		<tr>
			<td>Delay Heatstop t(H)</td>
			<td>050</td>
			<td>030</td>
			<td>050</td>
			<td>030</td>
		</tr>
		<tr>
			<td>Distance Heatstop s(H)</td>
			<td>030</td>
			<td>000</td>
			<td>030</td>
			<td>000</td>
		</tr>
		<tr>
			<td>Delay F(F1)</td>
			<td>000</td>
			<td>136</td>
			<td>000</td>
			<td>050</td>
		</tr>
		<tr>
			<td>Force Pull 1 F1</td>
			<td>000</td>
			<td>065</td>
			<td>200</td>
			<td>400</td>
		</tr>
		<tr>
			<td>Distance Pull 2 s(F2)</td>
			<td>000</td>
			<td>005</td>
			<td>000</td>
			<td>001</td>
		</tr>
		<tr>
			<td>Force Pull 2 F2</td>
			<td>000</td>
			<td>080</td>
			<td>000</td>
			<td>095</td>
		</tr>
		<tr>
			<td>Adjust (AD)</td>
			<td>121</td>
			<td>000</td>
			<td>121</td>
			<td>000</td>
		</tr>
	</tbody>
</table>
Table 2. Exemplarily puller protocol. For a horizontal puller (DMZ-Universal Puller, Zeitz-Instruments GmbH, Martinsried, Germany), using GB150F-8P filaments (Science Products, Hofheim, Germany) for both patch and iontophoretic pipettes.

Watch this Scientific Journal Video about Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration at JoVE.com

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

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 ...

fast-micro-iontophoresis-glutamate-gaba-useful-tool-to-investigate

Nanyang Technological University

Research

JoVE Journal

Neuroscience

19.4K Views.  University of Bonn. 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.

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

Bioengineering

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and &#947;-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and &#947;-aminobutyric acid receptors.

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal ...

Analyzing the Glutamate Release and Clearance at a Single Neuron Synapse in a Mouse Brain Slice

Source: Dvorzhak, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60113">Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice.</a> J. Vis. Exp. (2020)
This video demonstrates evaluating glutamate release and clearance at single corticostriatal synapses in a mouse brain slice. Electrical stimulation facilitates glutamate release at the synapse. The released glutamate alters sensor protein&#39;s fluorescence, which decays over time, indicating release and clearance efficiency.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: Recordings from Q175 wild-type ...

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

Summary

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Chapters in this video

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Analyzing the Glutamate Release and Clearance at a Single Neuron Synapse in a Mouse Brain Slice