The authors would like to thank the following organizations: National Natural Science Foundation of China (31171018, 31171355), the Science and Technology Division of Guangdong (2013KJCX0054), the Natural Science Foundation of Guangdong Province (2014A030313418, 2014A030313440), and Guangzhou Science and Technology Bureau (201607010320).

Animal housing and all animal experiments were conducted in accordance with procedures approved by the Ethics Committee for animal research at South China Normal University, according to the Guidelines for Animal Care established by the National Institute of Health.
1. Preparation of Solutions
<ol>
	<li>Prepare 500 mL of standard artificial cerebrospinal fluid (ACSF) containing the components described in our published work7 as listed in Table 1.
	<ol>
		<li>Use clean glass beakers with deionized water to prepare a fresh ACSF solution. Clean spatulas with deionized water and dry before using to add NaCl, KCl, NaH2PO4, NaHCO3, MgSO4, D-glucose, and add the CaCl2 after having bubbled the solution with 95% O2 / 5% CO2 mixture for ~20 min (as detailed in Table 1) to 500 mL of deionized water. Stir until fully dissolved.</li>
		<li>Ensure the ACSF is completely clear without any precipitation. Then adjust the pH to 7.2-7.4. Bubble with a gas mixture composed of 95% O2 and 5% CO2 for ~20 min.</li>
	</ol>
	</li>
	<li>Prepare 250 mL of slice cutting solution containing the chemicals7 described in Table 2.
	<ol>
		<li>Add KCl, MgCl2, NaH2PO4, NaHCO3, choline&#183;Cl, D-glucose and add the CaCl2 last as indicated in 1.1.1, to 250 mL of deionized water and dissolve as described in 1.1.1 and 1.1.2.</li>
		<li>After bubbling with 95% O2/5% CO2, place the solution in a freezer for 15-20 min so that the cutting solution becomes ice-cold and partially frozen. 
		NOTE: We use this cutting solution when making slices from 1-2 month-old mice; the formula may differ for mice older than 2 months.</li>
		<li>Prepare LPS solution by dissolving in sterile endotoxin-free isotonic saline (0.9% NaCl). LPS is administered intraperitoneally (0.5 mg/kg) and begin recording 2 h later.</li>
	</ol>
	</li>
</ol>
2. Preparation of Prefrontal Cortex Slice
<ol>
	<li>Take the cutting solution out of the freezer and homogenize to obtain a slush. Place the solution on ice and bubble constantly with 95% O2/5% CO2.</li>
	<li>Using large, sharp scissors, quickly decapitate a mouse as described9 and immediately put the head into a beaker filled with ice-cold (0-4&#176;C) cutting solution.</li>
	<li>Cut the top of the skull along the midline in a caudal-to-nasal direction with fine scissors and remove the lateral skull portions. Remove the brain with a fine spatula and drop into ice-cold cutting solution. 
	NOTE: Steps 2.2-2.3 must be done rapidly (ideally &#60; 1 min).</li>
	<li>Place the brain on the lid of a 9 cm Petri dish filled with ice. Rinse the brain with ice-cold cutting solution. Cut off the cerebellum and olfactory bulb with a razor blade.</li>
	<li>Apply cyanoacrylate glue to the specimen holder of a vibratome. Carefully place the brain block on the drop of glue, rostral side up, and immediately immerse in ice-cold cutting solution.</li>
	<li>Adjust the blade holder of the vibratome to an angle of 15&#176; with reference to the horizontal plane and prepare 350-&#181;m coronal brain slices (blade vibration frequency = 85 Hz, speed = 0.1 mm/s).</li>
	<li>Using a pipette, transfer the slices into the recovery chamber filled with ACSF, incubate for 1 h (at RT) with constant bubbling of 95% O2/5% CO2.</li>
</ol>
3. Whole-cell Patch Recording
<ol>
	<li>Make glass borosilicate micropipettes for use as recording electrodes or puff micropipettes according to the specific guidelines in the Puller Operation Manual. For recording electrodes, tip resistances are in the range 4-6 M&#8486;, while puff micropipettes have tip diameters in the range 2-5 &#181;m.</li>
	<li>Add Na+ channel blocker (TTX, 1 &#181;M), to block fast Na+&#160;channel and thus action potentials, to ACSF and maintain a constant flow rate of this solution of 2-3 mL/min in the recording chamber, by peristaltic pump into the chamber and aspiration out of it. Bubble the ACSF with 95% O2/5% CO2 constantly to ensure the viability of the slices.</li>
	<li>Transfer a brain slice into the recording chamber using a modified Pasteur pipette whose fine tip was cut to fit the slice size. Cover the slice with a platinum slice anchor with parallel nylon threads spaced &#8805;2 mm apart to hold the slice on the platform.</li>
	<li>Fill the recording micropipette with internal solution7 (Table 3), and fill the puff micropipettes with GABA at 10 &#181;M, 50 &#181;M, and 100 &#181;M (dissolved in ACSF), or ACSF as vehicle control.</li>
	<li>To prevent blockage with debris, apply light positive pressure using a 1 mL syringe before immersing the recording electrode in the ACSF. Using a micromanipulator under a microscope (4x objective lens), position the recording electrode and puff micropipette so that the tips appear in the center of the video image in the monitor (Figure 1A).</li>
	<li>Adjust the microscope focus (40x objective lens) while gradually lowering the puff micropipette and place it above the recording neuron at an angle of 45&#176;. Keep the distance between the tip of the puff micropipette and the neuron recorded between 20-40 &#181;m (Figure 1B). Slowly and carefully approach the neuron with the recording electrode and then release the positive pressure. Apply a weak and brief suction through the tubing connected to the electrode holder to create a giga-ohm seal.</li>
	<li>Maintain the voltage at 0 mV. After formation of the giga-ohm seal, compensate for fast and slow capacitance manually or automatically. Then apply a brief and strong suction through the tubing mentioned above to get into whole-cell mode.</li>
	<li>Record eIPSC in voltage-clamp mode. Apply low gas pressure (nitrogen, 4 - 6 psi) to the puff micropipette using a Picospritzer controlled by a Master 8 voltage step generator to deliver single or paired GABA puffs (Figure 2).</li>
	<li>Change the puff duration to obtain the best eIPSC results (Figure 3) and measure the eIPSC in the LPS-induced depression model ( Figure 4).</li>
</ol>

<ol>
	<li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres.">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> Nature. 260 (5554), 799-802 (1976).</li><li>Sakmann, B., Edwards, F., Konnerth, A., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+used+for+studying+synaptic+transmission+in+slices+of+mammalian+brain.">Patch clamp techniques used for studying synaptic transmission in slices of mammalian brain.</a> Q J Exp Physiol. 74 (7), 1107-1118 (1989).</li><li>Eyo, U. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+hyperactivity+recruits+microglial+processes+via+neuronal+NMDA+receptors+and+microglial+P2Y12+receptors+after+status+epilepticus.">Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus.</a> J Neurosci. 34 (32), 10528-10540 (2014).</li><li>Dickinson, G. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+Acts+through+Nicotinic+Receptors+to+Enhance+the+Firing+Rate+of+a+Subset+of+Hypocretin+Neurons+in+the+Mouse+Hypothalamus+through+Distinct+Presynaptic+and+Postsynaptic+Mechanisms.">Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms.</a> eNeuro. 2 (1), (2015).</li></ol>

The authors have nothing to disclose.

Puff application is widely used to evaluate postsynaptic receptor function3,4,7, but requires precise control in each experiment. We describe here a procedure involving whole-cell patch clamping, which demonstrates GABA-puff induced IPSCs (i.e., eIPSCs) in prefrontal cortical brain slices. The resistance of the recording electrode is about 5 M&#937;, while the tip diameter of puff micropipettes is about 2-5 &#181;m. Puff pressure is between 4-6 psi: this range is critical, as a higher pressure may cause neuronal damage, while a lower pressure can result in insufficient activation of the postsynaptic GABA receptors. The horizontal distance from the tip of the puff micropipette to the neuron recorded is about 20-40 &#181;m (the diameter of 1-2 neurons), and the angle between the puff micropipette and slice surface is about 45&#176;. The tip of the puff micropipette is vertically 20-40 &#181;m above the slice (z-axis). The puff duration should be in the range 10-150 ms. Under the above circumstances, puffs of 10-50 &#181;M GABA induce an appropriate neuronal response and the eIPSC amplitude and kinetic parameters obtained are reproducible. Although the protocol described here focuses on puff application to a slice patch, it is also suitable for recordings in neuronal cultures, as shown in our published work7.
We also show here eIPSCs resulting from paired-puff application of GABA. Paired-pulse protocols have been widely used to evaluate presynaptic properties7,11, but possible postsynaptic effects, such as changes in neurotransmitter-receptor binding rate and binding affinity, which could influence paired-pulse ratios, cannot be determined by presynaptic paired-pulse stimulation. The protocol described in this article provides a way to elucidate postsynaptic alterations in response to paired and even pulse-train stimuli. The reduction in eIPSC amplitude in the prefrontal cortex of LPS-injected mice suggests that altered levels or activity of GABA receptors might underlie LPS-induced behavioral changes.
Although, as with most drug delivery techniques, it is difficult to know the exact GABA concentration reaching the target cell, it is still possible to maintain the concentration of GABA at certain levels in the tissue by being consistent with the type of micropipettes used, and with parameters such as puff drive pressure and distance. In our case, the volume of solution puffed is about 1.1 &#181;L at 100 ms duration. Thus, puffing allows quantitative studies to be performed7,12.
The preparation of healthy brain slices from the acute prefrontal cortex is critical for the puff recording procedure we describe, and the following points should be borne in mind. First, dissect the brain and separate the prefrontal cortex quickly (&#60; 1 min) and then keep the brain block in ice-cold cutting solution for no more than 1 min. Next, tightly glue the brain block on top of the specimen holder of vibratome, or otherwise the brain block may float during sectioning. The speed and frequency of the vibratome should be set to generate even, healthy slices. Effort must be made to conduct all above procedures at 0-4 &#176;C to minimize neuronal excitability.
It is worth noting that the cutting solution we describe in this article should be used for 1-2 month-old mice. For older mice (i.e. &#62;3 months), the cutting solution should be replaced as described13. The puff technique is applicable to a range of patch-clamp experiments involving pharmacological treatments, and can also be used to test the effect of drugs on the electrical activity of any neurons.

The patch-clamp technique, a primary tool for investigating electrical signals in neurons, was developed in the 1970s1,2. A major advantage of this technique is that it provides knowledge on how specific treatments (e.g., pharmacological) may alter neuronal functions or channels in real time3. Pharmacological evaluation of neuronal function during whole-cell recording in a brain slice requires the application of drugs, such as agonists or antagonists of specific receptors, to the neurons being recorded. This method allows the identification of neuronal alterations that occur following application of a specific drug, thus leading to a better understanding of the physiological and pathological properties of the neurons4. Although pharmacological administration can be conducted via either perfusion5 or puff6, the latter is the superior technique. In particular: (i) puff application rapidly increases drug concentration around the recorded neuron to a level such that desensitization of the membrane receptors is prevented; (ii) the volume of drug puffed is extremely low, so that there is little effect on the bath solution, which therefore reduces any undesirable effects of the administered chemicals on brain slices; (iii) the puff protocol can be set and saved, making the experiment very precisely reproducible; (iv) puff application represents economical use of agonists/antagonists, particularly where such reagents are expensive or difficult to obtain.
Here, we will focus on recording whole-cell currents induced by puffing GABA in acutely prepared brain slices, a preparation that has the advantage of relatively well-preserved brain circuits. How we conduct puff-induced inhibitory currents7 will be described in this article. By using cesium (Cs+)-based internal solutions, and holding the neurons at 0 mV, we will introduce GABA-puff evoked inhibitory postsynaptic currents (eIPSCs) with appropriate technical details. Using a mouse model of depression induced by lipopolysaccharide (LPS) injection8, we show that the eIPSC amplitude evoked by a GABA puff is significantly reduced in slices of LPS-injected mice compared to vehicle controls. Our intention is that this article should show how the puff technique is widely applicable to studies aimed at evaluating the effect of any chemicals, compounds or drugs on neuronal activities in brain slices.

<table style="border-collapse:collapse;width:665pt" width="887">
	<colgroup>
		<col style="width:161pt" width="214" />
		<col style="width:140pt" width="187" />
		<col style="width:155pt" width="206" />
		<col style="width:210pt" width="280" />
	</colgroup>
	<tbody>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Glass Borosilicate micropipettes</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">BF150-86-10</td>
			<td class="xl18" style="width:210pt" width="280">1.50 mm outer diameter; 0.86 mm inner diameter</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Micropipette Puller</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">MODEL P-97&#160;</td>
			<td class="xl18" style="width:210pt" width="280">Flaming/Brow Micropipette Puller</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Micromanipulators</td>
			<td class="xl20" style="width:140pt" width="187">Shutter Instruments</td>
			<td class="xl20" style="width:155pt" width="206">MP-285</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Computer controlled Amplifier&#160;&#160;&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Molecular Devices</td>
			<td class="xl20" style="width:155pt" width="206">Multiclamp 700B</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Digital Acquisition system&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Molecular Devices</td>
			<td class="xl20" style="width:155pt" width="206">Digidata 1440A</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Imaging Camera</td>
			<td class="xl20" style="width:140pt" width="187">Nikon&#160;</td>
			<td class="xl18" style="width:155pt" width="206">2115001</td>
			<td class="xl18" style="width:210pt" width="280">Inspection equipment</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Microscopy&#160;</td>
			<td class="xl20" style="width:140pt" width="187">Nikon&#160;</td>
			<td class="xl20" style="width:155pt" width="206">Eclipse FN1&#160;</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Master 8</td>
			<td class="xl20" style="width:140pt" width="187">A.M.P.I.</td>
			<td class="xl20" style="width:155pt" width="206">Master-8 Pulse stimulator</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Vibratome Slicer</td>
			<td class="xl20" style="width:140pt" width="187">Leica</td>
			<td class="xl20" style="width:155pt" width="206">VT 1000S</td>
			<td class="xl18" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Picospritzer &#8546;</td>
			<td class="xl20" style="width:140pt" width="187">Parker Hannifin</td>
			<td class="xl20" style="width:155pt" width="206"></td>
			<td class="xl18" style="width:210pt" width="280">Pressure Systems for Ejection of 
			Picoliter Volumes in Cell Research</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Razor blade</td>
			<td class="xl20" style="width:140pt" width="187">Gillette</td>
			<td class="xl20" style="width:155pt" width="206">74-S</td>
			<td class="xl20" style="width:210pt" width="280">FLYING EAGLE</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video monitor</td>
			<td class="xl20" style="width:140pt" width="187">Panasonic</td>
			<td class="xl20" style="width:155pt" width="206">WV-BM 1410</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="214">502 Glue</td>
			<td class="xl20" style="width:140pt" width="187">Deli</td>
			<td class="xl18" style="width:155pt" width="206">7146</td>
			<td class="xl20" style="width:210pt" width="280">Cyanoacrylate Glue</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Peristaltic pump</td>
			<td class="xl20" style="width:140pt" width="187">Shanghai JIA PENG Corporation</td>
			<td class="xl20" style="width:155pt" width="206">BT100-1F</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video Camera</td>
			<td class="xl20" style="width:140pt" width="187">Olympus America Medical</td>
			<td class="xl20" style="width:155pt" width="206">OLY-150</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="214">Transfer Pipets</td>
			<td class="xl18" style="width:140pt" width="187">Biologix</td>
			<td class="xl18" style="width:155pt" width="206">30-0138A1</td>
			<td class="xl20" style="width:210pt" width="280"></td>
		</tr>
	</tbody>
</table>

whole-cell currents induced by puff application of gaba in brain slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

The puff technique allows researchers to study the effect of drugs on particular receptors on the surface of a given neuron. In this article, we focus on currents mediated by GABA receptors. Figure 1 shows the puff and recording micropipettes. To eliminate any effect on recorded neurons of the physical pressure generated by the puff, ACSF and sucrose puffs (Figure 2A) are used as controls. The response induced by a GABA puff (black) can be blocked by a GABA-receptor inhibitor, such as picrotoxin as reported10, while the baseline condition can be recovered by ACSF wash-out (blue), as shown in Figure 2B. Notably, neither an ACSF nor a sucrose puff induces a response, suggesting that responses induced by GABA puffs are physiological and are mediated by GABA receptors located on the surface of the recorded cell. The dose and puff-duration response curves are shown in Figure 3.
LPS has been shown to alter neuronal activities and animal behavior, but its effect on GABA receptor-mediated responses was not well known. When we compare currents induced by GABA puffs in brain slices of LPS-injected and vehicle control mice, we observe significantly reduced IPSC amplitudes due to LPS treatment (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56387/56387fig1.jpg" /> 
Figure 1. Diagram of the puff micropipette and recording electrode.&#160; 
(A) The distance between the puff and recording micropipettes. (B) The puff micropipette (left) and the recording electrode (right) on the surface of a brain slice.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56387/56387fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56387/56387fig2.jpg" /> 
Figure 2. Picrotoxin blockade of IPSC induced by GABA puff.&#160; 
(A)&#160;Puffing ACSF and sucrose (50 &#181;M) induces no response. (B) Puffing 50 &#181;M GABA induces an IPSC (black) (puff duration = 200 ms, 4-6 psi) in ACSF containing TTX (1 &#181;M), CNQX (20 &#181;M) and AP5 (50 &#181;M) to block action potentials and excitatory current mediated by AMPA and NMDA receptors. The GABA-induced IPSC is blocked by bath application of a GABA receptor inhibitor, 100 &#181;M picrotoxin (red). The IPSC recovers after wash-out of picrotoxin.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56387/56387fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56387/56387fig3.jpg" /> 
Figure 3. Dose and puff-duration response curves.&#160; 
(A) Sample IPSCs induced by 10, 50, and 100 &#181;M GABA (puff duration = 50 ms, 4-6 psi). (B) Puff-duration response curves. Black, n = 11; red, n = 8; blue, n = 10; polynomial fit. Data are shown as the means of multiple experiments (8 &#8804; n &#8804;11); error bars indicate SEM. (C) Repetitive currents evoked by GABA (50 &#181;M) puffs at inter-puff intervals of 1, 2 or 3 s (puff duration = 100 ms, 4-6 psi).&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56387/56387fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56387/56387fig4.jpg" /> 
Figure 4. eIPSC amplitude is reduced in LPS-injected mice compared to vehicle controls. 
Puffing GABA induces a significantly reduced IPSC amplitude in LPS-injected vs. control neurons (saline, 19.74 &#177; 1.93 pA/pF; LPS, 14.10 &#177; 1.30 pA/pF; p = 0.02).&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56387/56387fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration (mM)</td>
			<td>Weight (g/L)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.443</td>
			<td>119</td>
			<td>6.9547</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.551</td>
			<td>2.5</td>
			<td>0.1864</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>137.99</td>
			<td>1</td>
			<td>0.1380</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.007</td>
			<td>26.2</td>
			<td>2.2010</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>120.366</td>
			<td>1.3</td>
			<td>0.1565</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>198.17</td>
			<td>11</td>
			<td>2.1799</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>110.98</td>
			<td>2.5</td>
			<td>0.2775</td>
		</tr>
	</tbody>
</table>
Table 1. Recording ACSF. 
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration (mM)</td>
			<td>Weight (g/L)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.551</td>
			<td>2.5</td>
			<td>0.1864</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>95.211</td>
			<td>7</td>
			<td>0.6665</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>137.99</td>
			<td>1.25</td>
			<td>0.1725</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.007</td>
			<td>25</td>
			<td>2.1002</td>
		</tr>
		<tr>
			<td>Choline&#183;Cl</td>
			<td>139.63</td>
			<td>115</td>
			<td>16.0575</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>198.17</td>
			<td>7</td>
			<td>1.3872</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>110.98</td>
			<td>0.5</td>
			<td>0.0555</td>
		</tr>
	</tbody>
</table>
Table 2. Slice cutting solution.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Substance</td>
			<td>g/mol</td>
			<td>Concentration (mM)</td>
			<td>Weight (g/L)</td>
		</tr>
		<tr>
			<td>Cs gluconate</td>
			<td>328.052</td>
			<td>100</td>
			<td>32.8052</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>168.36</td>
			<td>5</td>
			<td>0.8418</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>238.301</td>
			<td>10</td>
			<td>2.3830</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>95.211</td>
			<td>2</td>
			<td>0.1904</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>110.98</td>
			<td>1</td>
			<td>0.1110</td>
		</tr>
		<tr>
			<td>BAPTA</td>
			<td>476.433</td>
			<td>11</td>
			<td>5.2408</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>551.1</td>
			<td>4</td>
			<td>2.2044</td>
		</tr>
		<tr>
			<td>Na2GTP</td>
			<td>523.2</td>
			<td>0.4</td>
			<td>0.2093</td>
		</tr>
	</tbody>
</table>
Table 3. Internal solution for whole-cell patch recordings.

Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

whole-cell-currents-induced-puff-application-gaba-brain

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.3K Views.  South China Normal University. We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Video: Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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.

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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

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

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...