Name: whole-cell currents induced by puff application of gaba in brain slices
Uploaded: 2017-10-12T14:00:00
Description: Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

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
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	<li>Prepare 500 mL of standard artificial cerebrospinal fluid (ACSF) containing the components described in our published work7 as listed in Table 1.
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		<li>Use clean gla...

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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 pressur...

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.

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			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Picospritzer &#8546;</td>
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			<td class="xl18" style="width:210pt" width="280">Pressure Systems for Ejection of 
			Picoliter Volumes in Cell Research</td>
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			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Razor blade</td>
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			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video monitor</td>
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			<td class="xl18" height="40" style="width:161pt;height:30.0pt" width="214">502 Glue</td>
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			<td class="xl18" style="width:155pt" width="206">7146</td>
			<td class="xl20" style="width:210pt" width="280">Cyanoacrylate Glue</td>
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			<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>
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			<td class="xl20" height="40" style="width:161pt;height:30.0pt" width="214">Video Camera</td>
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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...

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 ...

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