generating local cornu ammonis 1 (ca1) gamma oscillations using tetanic stimulations in a hippocampal brain slice

Generating Local Cornu Ammonis 1 (CA1) Gamma Oscillations Using Tetanic Stimulations in a Hippocampal Brain Slice

To mount the slice in the recording chamber, place a brain slice into a submerged recording chamber perfused with ACSF flowing at 1 to 2 milliliters per minute and heated to 32 degrees Celsius. The ACSF used for recordings differs from that in the holding chamber, as the magnesium concentration is increased from 2 millimolar to 4 millimolar.
Secure the slice by placing a semi-circular stainless steel weight on it with its strands parallel to hippocampal CA1. After that, increase the speed of perfusion to 8 to 10 milliliters per minute at 32 degrees Celsius.
Using a dissecting microscope, move the stimulating electrode to the middle of the stratum oriens. Then, move the recording electrode to the pyramidal layer as close to the stimulating electrode as possible. Subsequently, lower both the stimulating and recording electrodes 50 to 100 micrometers into the slice in order to generate the field EPSP of 1 millivolt in response to the 120 to 150 microamp test pulse.
To generate gamma oscillations, stimulate the tissue with a train of 20 times 0.1-millisecond pulses delivered at 200 hertz. This tetanic stimulus can yield reproducible responses when delivered every 5 minutes.

generating-local-cornu-ammonis-1-ca1-gamma-oscillations-using-tetanic

Universidad de Cartagena UDEC

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JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Setup for Cutting Brain Slices
<ol>
	<li>Prepare a cutting solution comprised of (mM) 125 Choline-Cl, 2.5 KCl, 0.4 CaCl2, 6 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 20 D-glucose saturated with carbogen gas (95% O2&#8211;5% CO2) and an artificial cerebrospinal fluid (aCSF) recording solution comprised of (mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 D-glucose, saturated with carbogen. Place the cutting solution on ice to keep it cold.</li>
	<li>Freeze approximately 400 ml of the cutting solution and blend together with 100 ml of unfrozen cutting solution to create an ice slurry. Bubble with carbogen (95 % O2&#8211;5 % CO2) at a flow of approximately 0.5 L/min through small caliber tubing or sintered glass to produce a steady but gentle flow of bubbles.</li>
	<li>Prepare a 250 ml beaker with a raised nylon mesh insert on which the brain slices will be placed. Fill with aCSF to cover the mesh by approximately 2 cm and bubble with carbogen, ensuring that the bubbles do not directly disrupt the slice holding area. It is also important that there are no air bubbles in the nylon mesh, so if any are present remove them. This will be the holding chamber and is kept at RT (20 - 25 &#176;C).</li>
	<li>Layout the dissecting instruments including a large pair of scissors, a small pair of scissors, small and large micro spatulas, and large and small pairs of forceps and chill them on ice. Place the vibratome tissue-cutting block on ice on a square of aluminum foil. Obtain 2 pieces of 6 cm filter paper, a single-edge razor blade, and a 25 ml beaker filled with the cutting solution slurry as well.</li>
	<li>Fill a second container with ice, and lay a piece of tissue paper on the ice and place a 12 cm culture dish on top. Fill the culture dish with cutting solution ice slurry and bubble with carbogen. This is the container in which the brain dissection with be performed.</li>
	<li>Prepare the vibratome. Remove a fresh double-edged razor blade (use a fresh blade every time) from its wrapping and spray with 80% ethanol then deionized water. Cut a 3 ml plastic transfer pipette at the point where it starts to taper. This will be used to transfer brain slices. Bend a 27 G needle at the base by approximately 45&#176; and attach to a 1 ml syringe. This will be used to manipulate slices during cutting.</li>
</ol>
2. Cutting Brain Slices
<ol>
	<li>Anesthetize a mouse (P16-P18) with 2% isoflurane or a locally approved method. Following induction, decapitate the animal with a large pair of scissors and drop the head into the 12 cm culture dish that contains bubbled cutting solution slurry. The slurry must totally immerse the head for rapid cooling.</li>
	<li>Hold the front of the head with one hand peeling the skin and connective tissue forward towards the nose. Using the small scissors cut the connective tissue to reveal the underlying skull. Then remove the muscles overlying the dorsal aspect of the skull and neck.</li>
	<li>Remove the brain from the skull by first securing the front of the skull with the large forceps and then make two lateral cuts through the bone either side of the foramen magnum using the small scissors (Figure 1, cuts labeled A1 and A2).
	<ol>
		<li>Make another cut between the eyes (just anterior of the bregma) (Figure 1, cut labeled B) then carefully cut along the sagittal suture anteriorly and reflect the cut skull sections to reveal the brain (Figure 1, cut labeled C).</li>
		<li>Use the small spatula to scoop out the brain and place on a culture dish. Be aware of the cranial nerves on the inferior aspect of the brain which will need to be severed, this can be done using the smaller sized micro spatula. Using the larger spatula transfer the brain to the 25 ml beaker filled with the cutting solution slurry.</li>
	</ol>
	</li>
	<li>Preparing the brain hemisphere for slicing.
	<ol>
		<li>Place one piece of the 6 cm filter paper in the bottom of a fresh culture dish then fill with fresh cutting solution slurry and bubble. Use the larger spatula to position the brain, ventral side down, onto the filter paper. Take a new and cleaned single-edge razor blade and cut the brain to remove the cerebellum, then make a cut along the midline to separate the brain into two hemispheres.</li>
	</ol>
	</li>
	<li>Preparing the vibratome tissue block.
	<ol>
		<li>Take the chilled vibratome tissue block, dry the surface, and place a drop of cyanoacrylate glue in the middle, and spread evenly to the approximate size of the brain. Use the small spatula to manipulate one of the brain hemispheres onto the large micro spatula so the medial side of the brain is down.</li>
		<li>Touch the edge of the spatula at the interface of the brain on the second piece of filter paper to remove as much of the solution as possible. Slide the brain off the larger spatula using the smaller spatula to guide it onto the glue.</li>
		<li>Secure the cutting block into the vibratome chamber and fill the chamber with cutting solution slurry and bubble, ensuring the brain is completely immersed. Rotate the cutting block so the ventral side of the brain is facing the blade.</li>
	</ol>
	</li>
	<li>Slicing the brain. 
	&#160;NOTE: Each vibratome is unique so follow the manufacturer&#39;s instructions.
	<ol>
		<li>Set the slice thickness to 450 &#181;m, and ensure the blade is vibrating as it moves through the brain at a speed of approximately 0.3 mm/s. Cut entirely through the brain from the ventral side to the cortical surface, this will produce whole brain sagittal slices that can be used for electrophysiological recordings. Slices will be produced laterally to medially and typically 3 - 4 slices can be cut from each hemisphere.</li>
		<li>If the base of the slice lifts, use the bent 27 G needle to gently press the slice back down. As each slice is cut, use the transfer pipette to move it onto the platform in the holding chamber where they are viable for up to 8 hrs.</li>
	</ol>
	</li>
</ol>
3. Extracellular Electrophysiology Recordings
<ol>
	<li>Mounting the slice in the recording chamber.
	<ol>
		<li>Using the transfer pipette, place a brain slice into a submerged recording chamber perfused with aCSF flowing at 1 - 2 ml/min and heated to 32 &#176;C. The aCSF used for recordings differs from that in the holding chamber as the Mg2+ concentration is increased from 2 mM to 4 mM.</li>
		<li>Secure the slice with a &#34;harp&#34; (semi-circular stainless steel with nylon strands stretched across at 2 - 3 mm spacing). Place the harp so that the strands run parallel to CA1. Increase the speed of the perfusion to 8 - 10 ml/min at 32 &#176;C.</li>
	</ol>
	</li>
	<li>Under a dissecting microscope place a stimulating electrode and a recording electrode (glass electrode filled with the aCSF recording solution) on the surface of the stratum radiatum of the CA1 (Figure 2A). Place the stimulating electrode first, then place the recording electrode.</li>
	<li>Stimulate the Schaffer collaterals with a 120 - 150 &#181;A amplitude and 0.1 msec duration test pulse and observe the resulting field excitatory post synaptic potential (fEPSP) waveform to determine slice health (Figure 2B). The stimulating and recording electrodes may need to be moved into the slice approximately 50 - 100 &#181;m from the slice surface to get a fEPSP recording with a small fiber volley and large amplitude. The Schaffer collateral evoked fEPSPs are stereotypical and provide a good indicator of slice health. Healthy slices typically show a fiber volley to fEPSP amplitude ratio of less than 0.3.</li>
	<li>Repositioning the electrodes.
	<ol>
		<li>Using a dissecting microscope, move the stimulating electrode to the middle of the stratum oriens and move the recording electrode to the pyramidal cell layer as close to the recording electrode as possible, as shown in Figure 3A. The stimulating and recording electrodes may need to be pushed into the slice approximately 50 - 100 &#181;m so that the fEPSP response amplitude to the 120 - 150 &#181;A test pulse is approximately 1 mV.</li>
	</ol>
	</li>
	<li>Generating &#947; oscillations.
	<ol>
		<li>To generate &#947; oscillations, stimulate the tissue with a train of 20 x 0.1 msec pulses delivered at 200 Hz. This tetanic stimulus can yield reproducible responses when delivered every 5 min.</li>
	</ol>
	</li>
	<li>Use the following recording parameters.
	<ol>
		<li>Scale the gain of the output signal to match the input voltage range of the analog-to-digital converter. Make sure that the maximum expected signal excursion uses a minimum of 30% of the input voltage range. Be careful when setting gains too high, as signals might clip. The typical bandwidth needed for field recordings is 500 Hz with AC coupling at 0.1 Hz to remove baseline drift.</li>
		<li>Digitize at least 4 - 5 times faster than the corner frequency of the low pass filter to avoid signal aliasing.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-(N-Ethyl-N-phenylamino)-1,2 dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288)</td>
			<td>Sigma-Aldrich</td>
			<td>Z3777</td>
			<td></td>
		</tr>
		<tr>
			<td>Biuculline</td>
			<td>Sigma-Aldrich</td>
			<td>14340</td>
			<td></td>
		</tr>
		<tr>
			<td>6-cyano-7-nitroquinoxa- line-2,3 dione (CNQX)</td>
			<td>Sigma-Aldrich</td>
			<td>C127</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel</td>
			<td>Sigma-Aldrich</td>
			<td>266965</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbamazepine</td>
			<td>Sigma-Aldrich</td>
			<td>C4024</td>
			<td></td>
		</tr>
		<tr>
			<td>(2R)-amino-5-phosphonopentano ate (APV)</td>
			<td>Tocris Bioscience</td>
			<td>105</td>
			<td></td>
		</tr>
		<tr>
			<td>Retigabine</td>
			<td>ChemPacific</td>
			<td>150812-12-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline-Cl</td>
			<td>Sigma-Aldrich</td>
			<td>C1879-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S9638-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2.2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>223506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M2670-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode glass</td>
			<td>Harvard Apparatus</td>
			<td>GC150F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentric bipolar stimulating metal electrode</td>
			<td>FHC</td>
			<td>CBBPF75</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Isolator</td>
			<td>Getting Instruments</td>
			<td>Model BJN8-9V1</td>
			<td></td>
		</tr>
		<tr>
			<td>Model 1800 amplifier</td>
			<td>A-M systems</td>
			<td>Model 1800 amplifier</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>National Intruments</td>
			<td>NI USB-6211</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica</td>
			<td>VT1200s</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Hatch, R. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52877">Generation of Local Ca1 &#947; Oscillations by Tetanic Stimulation.</a> J. Vis. Exp. (2015).
This video demonstrates the procedure of inducing local CA1 gamma oscillations in a CA1 hippocampal mouse brain slice using tetanic simulations. This method is important to understand the neuronal network activity.

<img alt="Figure 1" src="/files/ftp_upload/22866/22866_Figure1.jpg" /> 
Figure 1. Schematic of the skull and overlying skin showing the location of cuts to reveal the brain. A1, A2, B and C mark the locations of cuts that need to be made to open up the skull for the removal of the brain.
<img alt="Figure 2" src="/files/ftp_upload/22866/22866_Figure2.jpg" /> 
Figure 2. Placement of the stimulati...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Setup ...

Secure a brain slice in a recording chamber with a continuous perfusion of aCSF containing high magnesium that blocks N-methyl-D-aspartate or NMDA glutamate receptors. 
Position the stimulating and recording electrodes in the hippocampal Cornu ammonis 1 or CA1 stratum radiatum. 
Stimulate to obtain an electrical signal, confirming intact neuronal connections.
Reposition the stimulating electrode to the stratum oriens and the recording electrode to the stratum pyramidale, then insert them deeper.
Apply tetanic stimulations, high-frequency electrical pulses to induce excitatory neurotransmitter glutamate release from Schaffer collateral axons.
Glutamate binds to glutamate receptors on CA1 neurons, facilitating sodium influx and membrane depolarization.
This unblocks NMDA receptors, allowing sodium and calcium influx and enhancing neuron excitation.
The excited interneurons in the stratum oriens release the inhibitory neurotransmitter GABA, interacting with CA1 neurons.
This balance of excitatory and inhibitory inputs leads to synchronized controlled excitation of the CA1 neuronal network, generating rhythmic brain waves called gamma oscillations, crucial for attention and memory.

20 조회수. 해마 뇌 절편에서 테타닉 자극을 사용하여 국소 cornu ammonis 1 (CA1) 감마 진동 생성

비디오: 해마 뇌 절편에서 테타닉 자극을 사용하여 국소 cornu ammonis 1 (CA1) 감마 진동 생성 - 실험

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

행동 하는 쥐의 해 마에서 진동 제한 공간 기록

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

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JoVE Journal

신경과학

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.

전기 생리학 필드 기록에 의하여 인터라멜라 해마 CA1에 있는 장기 시냅스 가소성 조사

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

급성 마우스 뇌 슬라이스 자발적인 해 마 네트워크 활동을 조사

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Generating Local Cornu Ammonis 1 (CA1) Gamma Oscillations Using Tetanic Stimulations in a Hippocampal Brain Slice

내레이션 대본

Generating Local Cornu Ammonis 1 (CA1) Gamma Oscillations Using Tetanic Stimulations in a Hippocampal Brain Slice