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. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+determining+the+recruitment+of+inositol+trisphosphate+receptor+channels+during+calcium+puffs.">Factors determining the recruitment of inositol trisphosphate receptor channels during calcium puffs.</a> Biophys J. 105 (11), 2474-2484 (2013).</li><li>Lee, J., 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=Columnar+distribution+of+activity+dependent+gabaergic+depolarization+in+sensorimotor+cortical+neurons.">Columnar distribution of activity dependent gabaergic depolarization in sensorimotor cortical neurons.</a> Mol Brain. 5, 33 (2012).</li><li>Glykys, J., Mody, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+main+source+of+ambient+GABA+responsible+for+tonic+inhibition+in+the+mouse+hippocampus.">The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus.</a> J Physiol. 582, 1163-1178 (2007).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+modulates+KCC2+expression+and+function+in+hippocampal+GABAergic+inhibition.">APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.</a> Elife. 6, (2017).</li><li>Dunn, A. J., Swiergiel, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+interleukin-1+and+endotoxin+in+the+forced+swim+and+tail+suspension+tests+in+mice.">Effects of interleukin-1 and endotoxin in the forced swim and tail suspension tests in mice.</a> Pharmacol Biochem Behav. 81 (3), 688-693 (2005).</li><li>Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+Patch-clamp+Recordings+from+the+Somatodendritic+Domain+of+Nigral+Dopamine+Neurons.">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J Vis Exp. (117), (2016).</li><li>Wondolowski, J., Frerking, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-dependent+postsynaptic+expression+of+kainate+receptors+on+hippocampal+interneurons+in+area+CA1.">Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1.</a> J Neurosci. 29 (2), 563-574 (2009).</li><li>Yang, L., Wang, Z., Wang, B., Justice, N. J., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+regulates+Cav1.2+L-type+calcium+channel+levels+and+function+to+influence+GABAergic+short-term+plasticity.">Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity.</a> J Neurosci. 29 (50), 15660-15668 (2009).</li><li>Huo, Q., 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=Prefrontal+Cortical+GABAergic+Dysfunction+Contributes+to+Aberrant+UP-State+Duration+in+APP+Knockout+Mice.">Prefrontal Cortical GABAergic Dysfunction Contributes to Aberrant UP-State Duration in APP Knockout Mice.</a> Cereb Cortex. , (2016).</li><li>Zhou, W. L., Gao, X. B., Picciotto, M. 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><tbody><tr><td>Glass Borosilicate micropipettes</td><td>Shutter Instruments</td><td>BF150-86-10</td><td>1.50 mm outer diameter; 0.86 mm inner diameter</td></tr><tr><td>Micropipette Puller</td><td>Shutter Instruments</td><td>MODEL P-97&amp;nbsp;</td><td>Flaming/Brow Micropipette Puller</td></tr><tr><td>Micromanipulators</td><td>Shutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system&amp;nbsp;</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Imaging Camera</td><td>Nikon&amp;nbsp;</td><td>2115001</td><td>Inspection equipment</td></tr><tr><td>Microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Eclipse FN1&amp;nbsp;</td><td></td></tr><tr><td>Master 8</td><td>A.M.P.I.</td><td>Master-8 Pulse stimulator</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica</td><td>VT 1000S</td><td></td></tr><tr><td>Razor blade</td><td>Gillette</td><td>74-S</td><td>FLYING EAGLE</td></tr><tr><td>Video monitor</td><td>Panasonic</td><td>WV-BM 1410</td><td></td></tr><tr><td>502 Glue</td><td>Deli</td><td>7146</td><td>Cyanoacrylate Glue</td></tr><tr><td>Peristaltic pump</td><td>Shanghai JIA PENG Corporation</td><td>BT100-1F</td><td></td></tr><tr><td>Video Camera</td><td>Olympus America Medical</td><td>OLY-150</td><td></td></tr><tr><td>Transfer Pipets</td><td>Biologix</td><td>30-0138A1</td><td></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/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/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/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/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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.3K Views.  South China Normal University. We beschrijven de bladerdeeg-techniek, waarbij farmacologische reagentia tijdens geheel-cel patch-clamp opname kunnen worden beheerd, en benadrukken verschillende aspecten van de functies die cruciaal voor het succes ervan zijn.

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, ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

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 ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Source: Feng, Y. et al., <a href="https://app.jove.com/v/56387">Whole-cell Currents Induced by Puff Application of GABA in Brain Slices.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the recording of whole-cell currents induced by gamma-aminobutyric acid, or GABA, in a mouse prefrontal cortical brain slice. Controlled delivery of GABA opens chloride channels in a postsynaptic neuron, generating inhibitory postsynaptic currents, or IPSCs. These IPSCs increase with higher GABA concentrations, reflecting GABA&#39;s inhibitory activity on synaptic transmission.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Solutions

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

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