recording ca3-ca1 synaptic responses in a rat hippocampal slice

Recording CA3-CA1 Synaptic Responses in a Rat Hippocampal Slice

In synaptic tagging and capture experiments, under the microscope, position the two stimulating electrodes in the stratum radiatum of the CA1 region to stimulate the Schaffer collateral fibers and one recording electrode in the apical dendritic region of CA1 midway between the stimulating electrodes to record fEPSP responses.
Place another recording electrode in the stratum pyramidale layer for recording population spike. When all the electrodes touch the slice, deliver a test stimulation to ensure a proper field EPSP signal can be obtained in both the inputs.
Once a proper fEPSP signal is obtained, carefully, lower the electrodes about 200 microns further using the fine movement knobs of the manipulators, then, allow 20 minutes for the slice to recover. After that, determine the input/output relation by measuring the slope of field EPSP upon a range of current intensities from 20 microamps to 100 microamps.
Then, for each input, set the stimulation intensity that evokes 40% of the maximum field EPSP slope as the constant stimuli throughout the experiment. After 15 to 20 minutes, start recording the baseline.
Record a stable baseline of at least 30 to 60 minutes before LTP/LTD induction.

recording-ca3-ca1-synaptic-responses-in-a-rat-hippocampal-slice

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Electrophysiology Techniques

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Recording of CA3-CA1 Synaptic Responses
NOTE: The electrophysiology set-up used for field potential recording is shown in&#160;Figure 1A. A Faraday cage is strongly recommended if the electrical interference is beyond the control after the proper grounding of the electrical settings. Many different types of submerged and interface chambers are commercially available. However, interface chambers are preferred as slices exhibit more robust synaptic responses in them.
<ol>
	<li>Positioning of electrodes&#8203;
	<ol>
		<li>Turn on the electrical apparatus (stimulators and amplifiers) to be used. Mount and secure the stimulating and recording electrodes in the plexiglass holders of the micromanipulators. &#160; 
		NOTE: We use monopolar, lacquer-coated, stainless-steel electrodes of 5 M&#937; resistance for both stimulating and recording purposes.</li>
		<li>Before use, insert these electrodes inside the pulled glass capillaries and secure with epoxy glue, exposing only small portion of the electrode tip (Figure 1E). This gives strength to the otherwise slender electrodes and helps to secure them firmly in the electrode holders.</li>
		<li>Guided under the microscope, position the stimulating electrode(s) in the stratum radiatum of the CA1 region to stimulate the Schaffer collateral fibers and the recording electrode in the apical dendritic region of CA1 to record field-EPSP (fEPSP) responses. &#160; 
		NOTE: Approaching the liquid surface above the slice with the electrodes gives a sound that helps to locate the surface of the slice quickly (provided, the amplifier is connected to a loudspeaker).</li>
		<li>In synaptic tagging and capture experiments, according to the need of the experiment, position two or three stimulating electrodes (S1, S2, or S3) on either side of the recording electrode to stimulate two or more independent but overlapping inputs. Position the stimulating and recording electrodes about 200 &#181;m apart.</li>
		<li>If necessary, locate another recording electrode in the stratum pyramidale layer for recording population spike (Figure 2A). When both electrodes have touched the slice, using the acquisition software, give a test stimulation to ensure a proper fEPSP signal. &#160; 
		NOTE: We use biphasic, constant-current pulses (impulse duration 0.1 msec/half-wave) for test stimulation.</li>
		<li>Once a proper fEPSP signal is obtained, carefully lower the electrodes about 200 &#181;m deep using fine movement knobs of the manipulators. Allow 20 min&#160;for the slices to recover.</li>
	</ol>
	</li>
	<li>Input-output relation&#8203;
	<ol>
		<li>Determine the input-output relation (afferent stimulation vs fEPSP slope) for each input by measuring the slope value at a range of current intensities. Perform this between 20 &#181;A to 100 &#181;A. Then set the stimulation intensity for each input to obtain 40% of the maximum fEPSP slope. Keep this constant throughout the experiment.</li>
		<li>After 15-20 min, start recording the baseline. Monitor the fEPSP slope closely during this period and reset the stimulus intensity if the slope fluctuates more than 10% from the set value and start a new baseline. Record at least 30 min&#160;or 1 hr stable baseline before proceeding. &#160; 
		NOTE: For the test or the baseline stimulation, we use four sweeps of 0.2 Hz biphasic, constant current pulses (0.1 msec per polarity) given every 5 min. An average slope of these four responses is then considered as one repeat. The signals are filtered and amplified by a differential amplifier, digitized using an analog-to-digital converter and monitored online with custom-made software.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>I. ACSF component chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1. Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Magnesium sulphate heptahydrate (MgSO4.7H20)</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Calcium chloride dihydrate (CaCl2.2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>5. Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>6. Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>7. D-Glucose anhydrous (C6H12O6)</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>II. Electrophysiology Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1. Microscope</td>
			<td>Olympus, Japan</td>
			<td>Model SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Temperature Controller</td>
			<td>Scientific Systems Design Inc. Canada</td>
			<td>PTC03</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Differential AC Amplifier</td>
			<td>AM Systems, USA</td>
			<td>Model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>4. Isolated Pulse Stimulator</td>
			<td>AM Systems, USA</td>
			<td>Model 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>5. Oscilloscope</td>
			<td>Rhode &#38; Schwarz</td>
			<td>HM0722</td>
			<td></td>
		</tr>
		<tr>
			<td>6. Digital-Analog Converter</td>
			<td>Cambridge Electronic Design Ltd. Cambridge, UK</td>
			<td>CED-Power 1401-3</td>
			<td></td>
		</tr>
		<tr>
			<td>7. Interface Brain Slice Chamber</td>
			<td>Scientific Systems Design Inc. Canada</td>
			<td>BSC01</td>
			<td></td>
		</tr>
		<tr>
			<td>8. Tubing Pump</td>
			<td>Ismatec, Idex Health &#38; Science, Germany</td>
			<td>REGLO-Analog</td>
			<td></td>
		</tr>
		<tr>
			<td>9. Carbogen Flowmeter</td>
			<td>Cole-Parmer</td>
			<td>03220-44</td>
			<td></td>
		</tr>
		<tr>
			<td>10. Fiber Light Illuminator</td>
			<td>Dolan-Jenner Industries</td>
			<td>Fiber Lite MI-150</td>
			<td></td>
		</tr>
		<tr>
			<td>11. Micromanipulators</td>
			<td>Marzhauser Wetzlar, Germany</td>
			<td>00-42-101-0000 (MM-33)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>00-42-102-0000 (MM-32)</td>
			<td></td>
		</tr>
		<tr>
			<td>12. Electrodes</td>
			<td>AM Systems, USA</td>
			<td>571000 (Stainless steel; 0.010, 5M&#937;, 8 degree)</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Shetty, M. S. et al., <a href="https://app.jove.com/v/53008">Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a method to record CA3-CA1 synaptic responses in a rat hippocampal slice. It outlines the steps involved in placing the stimulating and recording electrodes on the hippocampal slice and measuring the field excitatory postsynaptic potential (fEPSP) to assess synaptic response strength.

<img alt="Figure 1" src="/files/ftp_upload/22871/22871_Figure1.jpg" /> 
Figure 1.&#160;Electrophysiology set-up for field-potential recordings consisting of&#160;(A)&#160;stimulators (b) a differential amplifier (c) an analog-to-digital converter (d) Oscilloscope (e) computer with acquisition software (f) Vibration-resistant table-top (g) microscope with &#62;4x magnification (h) interface brain-slice chamber (i) a perfusion system for ACSF and carbo...

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Recording of CA3-CA1 ...

Place a rat hippocampal slice in an electrophysiology setup. 
Position the stimulating electrodes in the stratum radiatum layer of the hippocampal CA1 region, which receives axonal projections from the CA3 neurons.
Place a recording electrode near the CA1 apical dendrites.
Position another recording electrode in the stratum pyramidale layer.
Upon stimulation, the CA3 axonal projections depolarize and generate action potentials, releasing excitatory neurotransmitters.
These neurotransmitters bind to postsynaptic CA1 apical dendrites, inducing a positive ion influx and membrane depolarization, termed excitatory postsynaptic potential, or EPSP.
The recording electrode measures summed EPSPs from the apical dendrites &mdash; the field EPSP or fEPSP. Once stable fEPSP signals are obtained, lower the electrodes and pause briefly.
Apply a range of stimulation intensities to determine the input-output relationship with the fEPSP slope, then select and apply a specific intensity based on the slope.
The second recording electrode measures the combined action potentials from CA1 neuronal cell bodies, generating population spikes.

27 次观看. 在大鼠海马切片中记录 CA3-CA1 突触反应

视频: 在大鼠海马切片中记录 CA3-CA1 突触反应 - 实验

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

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神经科学

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

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In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Recording CA3-CA1 Synaptic Responses in a Rat Hippocampal Slice

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Recording CA3-CA1 Synaptic Responses in a Rat Hippocampal Slice