eoe sub articles

EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
1. Acute Hippocampus In Vitro Preparation
NOTE: Isolating the intact hippocampal preparation involves three major steps: (1) Preparation of solutions and equipment, (2) Dissection of the hippocampus and (3) Setting up the fast perfusion rate system necessary for generation of intrinsic theta oscillations. In this protocol, the timely performance of procedures &#8211; from dissection to recording &#8211; is particularly important because the isolated hippocampus constitutes such a dense but delicate preparation that maintaining functional connectivity of the structure in vitro requires great care. Preparing everything beforehand ensures that an adequate level of perfusion is available as early as possible to minimize cell damage and maintain physiological function.
<ol>
	<li>Sucrose-based artificial Cerebral Spinal Fluid (aCSF) solution
	<ol>
		<li>Prepare 1X stock sucrose solution to be used for dissection and post-dissection incubation. Combine all components listed in Table 1, except for CaCl2, into 1 L of deionized water and store at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Standard aCSF solution
	<ol>
		<li>Prepare standard aCSF for high flow rate perfusion of the isolated hippocampus during electrophysiological recordings. To make the concentrated (5X) stock aCSF, dissolve all the components listed in Table 2, except CaCl2, in 2 L of deionized water and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the equipment
	<ol>
		<li>Before starting dissection, set up the bench area with an ice-filled plastic tray (30 x 20 x 5 cm), carbogen-connected tubing lines, and three glass Petri dishes (10 x 2 cm) (See Figure 1).</li>
		<li>Add 300 mL of sucrose solution (1X stock) to a 500 mL beaker, bubble it with carbogen (95% O2, 5% CO2) and add Ca2+ [1.2 mM].</li>
		<li>Transfer 60 mL of this sucrose solution to a glass Petri dish and leave at room temperature. Place the remainder in a 4 &#176;C freezer for 10 - 15 min.</li>
		<li>Bubble the ice-cold sucrose solution with carbogen throughout the dissection.</li>
		<li>Fill a second Petri dish (cold holding chamber) with sucrose solution (4 &#176;C) and keep it on ice.</li>
		<li>Add 30 mL of ice-cold sucrose solution to a 50 mL beaker.</li>
	</ol>
	</li>
</ol>
2. Whole Hippocampus Dissection
NOTE: The method for dissecting the isolated hippocampus is essentially identical to the one developed and described originally but with additional details and changes regarding the perfusion rate and recording techniques.
<ol>
	<li>Brain dissection
	<ol>
		<li>Anesthetize mouse under a fume hood with a small paper towel soaked with 1 mL of isoflurane (100%) that is added into the cage.</li>
		<li>Decapitate the anesthetized mouse and quickly submerge the head in ice-cold sucrose solution (50 mL beaker, ~ 5 s). Transfer onto a paper towel.</li>
		<li>Use a scalpel to make a medial skin incision (caudal to rostral) and push the skin on the sides to expose the cranium.</li>
		<li>Cut open the skull with a small scissors and use a spatula to rapidly extract the brain and drop it into the Petri dish containing ice-cold sucrose solution. Allow 1-2 min for the brain to cool.</li>
		<li>While the brain is recovering, add 2 - 3 mL of sucrose solution onto a lens paper-covered Petri (dissection) dish on ice.</li>
	</ol>
	</li>
	<li>Isolating the hippocampus
	<ol>
		<li>Place the brain onto the dissection dish in an upright position.</li>
		<li>Remove the cerebellum and frontal cortex with a razor blade. Cut the brain in half along the midsagittal plane and return the two isolated hemispheres to the holding chamber. Allow a further 1 - 2 min for recovery.</li>
		<li>Place a single hemisected brain upright onto the dissection dish.</li>
		<li>Rotate the dish until midsagittal structures face the observer and the embedded outline of the septal complex is visible as a thin pear-shaped layer of tissue anterior to the thalamus.</li>
		<li>Hold the hemisected brain steady with the micro spatula and place the small end of the polytetrafluoroethylene (PTFE)-coated spatula immediately behind (caudal to) the septal area.</li>
		<li>Insert the coated spatula beneath the septum and move the tip down until the dissection dish is reached. Sever the fibers that connect the septal area caudally. Repeat the same operation along the anterior edge of the septum and cut the fibers that connect to the frontal part of the brain.</li>
		<li>With the spatula lightly holding the inner part of the cortex just above the hippocampus in an upright position, use the micro spatula to carefully pull down the thalamic, hypothalamic, and remaining brain stem nuclei. Then, use the spatula to cut off and remove the pulled-away tissue.</li>
		<li>Turn the isolated hemisphere on its side and identify the outline of the hippocampus.</li>
		<li>Insert the coated spatula in the lateral ventricle underneath the rostral end of the dorsal hippocampus.</li>
		<li>Hold the spatula horizontally aligned with the midsagittal plane of the hemisected brain and slide it through the smooth contour of ventricular walls until the tip emerges caudally.</li>
		<li>Hold the spatula beneath the hippocampus and lightly press it onto the connecting fibers along the inside layer where the hippocampus joins with the overlying cortex. Apply the micro spatula on the external side of this layer and slide against the coated spatula to slice through the connecting fibers.</li>
		<li>To complete the extraction, rotate the dissection dish and insert the coated spatula beneath the ventral hippocampus. Lightly hold the inside of the cortical fold with the spatula and cut through the entorhinal connections using a slicing motion against the micro spatula. 
		NOTE: The whole septohippocampal complex can be removed by placing the coated spatula under the entire hippocampus, and pulling out the remaining brain tissue underneath.</li>
		<li>Once isolation is complete, keep the hippocampus resting on the dish and add a drop of ice-cold sucrose solution to keep it cool. Carefully trim off any remaining cortex and fibers and separate the hippocampus from the septum by gently applying a razor blade through the fornix. 
		NOTE: If patch clamp recordings are to be performed from pyramidal cells, the isolated hippocampus can be transversally cut at a 45&#176; angle to expose the pyramidal layer of Cornu Ammonis (CA)1 (&#34;angle-cut&#34; preparation) without compromising the local network circuitry in the remaining portion of the hippocampus.</li>
		<li>Transfer the preparation to room temperature sucrose solution and let it recover for 15 - 30 min before transferring to the recording chamber.</li>
	</ol>
	</li>
</ol>
3. Set Up the Fast Perfusion for Recording the Isolated Hippocampus
<ol>
	<li>Set up the gravity-fed perfusion system to enable continuous high-speed inflow of oxygenated aCSF at 20 - 25 mL/min.
	<ol>
		<li>Prepare aCSF (1X) flasks in advance and immerse them in a warming bath (~30 &#176;C) at least 15 min before start of experiment. Turn on the in-line solution heater system (30 &#176;C).</li>
		<li>Use aquarium bubblers and tubing lines connected to a carbogen tank to oxygenate aCSF throughout the experiment, and add CaCl2 [2 mM] prior to the start of perfusion.</li>
		<li>Stop aCSF flow and transfer the hippocampus to the recording chamber using the wide end of a glass pipette. Allow the sucrose saturated preparation to sink and settle at the bottom.</li>
		<li>Place the preparation in the center of the recording chamber (in line with the inlet-outlet axis) with the smooth surface of CA1 and SUB on top.</li> 
 <li> Stabilize the hippocampus with small weights at the septal and temporal extremities and restart aCSF flow.</li>
	</ol>
	</li>
</ol>
4. Electrophysiology in the Isolated Hippocampus
<ol>
	<li>Extracellular recording of in vitro hippocampal theta oscillations
	<ol>
		<li>For extracellular monitoring of Local Field Potential (LFP) or single-unit activity from the in vitro isolated hippocampus, use glass micropipettes (1 - 3 M&#937;) filled with aCSF. Record low-noise AC-coupled field potential signals with a patch clamp amplifier of gain x1000, online band-pass filtering 0.1 - 500 Hz, and a sampling rate of 5-20 kHz. 
		NOTE: The custom-built microscope used in this protocol is equipped with low- (2.5X) and high-magnification (40X) objectives for a low-magnification view of the hippocampus, as well as infrared video microscopy and epifluorescence for single-cell recognition. The components of this system include the upright fluorescence microscope, fluorescence filter cubes, an analog video camera and digital frame grabber with imaging software, an on-stage custom perfusion chamber (Figure 2A, inset i), and high-precision micromanipulators for neuronal recordings and optic fiber placement.</li>
		<li>Use the camera mounted on the microscope and connected to a computer display to inspect the hippocampal preparation under low magnification (2.5X) and rapidly check that there are no undercuts or damage to the external surface. The diagonally oriented arrangement of alveus fibres along the smooth surface of CA1 should be visible (Figure 2A, inset ii) when shining transmitted light through the hippocampus.</li>
		<li>Use the low-magnification wide-field objective to view the isolated hippocampus and the placement of LFP electrodes in specific recording locations. 
		NOTE: A layout of the isolated hippocampal preparation with multiple electrodes placed in different recording locations is illustrated in Figure 2A.</li>
		<li>Lower an LFP electrode into the bath until it touches the surface (alveus) of the CA1 area. 
		NOTE: This usually produces a fast transient in the AC-coupled recording, similar to what happens when the electrode comes in contact with the recording media. An audio monitor can be useful to listen to the LFP channel signal after this step. 
		NOTE: Often, early signs of activity will only appear after 10 - 15 min, so it is important not to advance quickly into the preparation. If after this period, the surface LFP electrode is still not picking up activity, advance a little further down through the stratum oriens and pause periodically to see if any form of activity arises while approaching the principal cell layer. If nothing is detected after a further 5 - 10 min, carefully retract the electrode and place it elsewhere along the same region.</li>
		<li>Advance the LFP electrode through the pyramidal layer. Observe that extracellular spiking activity initially increases as single-unit discharge from individual neurons (mostly pyramidal and inhibitory basket cells) is detected. Lower the electrode further and note that spiking begins to fade again as the tip crosses into the radiatum. Observe that a clearly visible network oscillation in the theta-frequency range (4 - 12 Hz) becomes apparent and reaches maximum amplitude (~100 - 300 &#181;V) as the recording location is lowered through the radiatum.</li>
	</ol>
	</li>
	<li>Theta oscillations across a single region
	<ol>
		<li>To test the spatial properties of spontaneous theta oscillations across the CA1 region, place the tip of a reference LFP electrode in a medial CA1 site (medially located along the longitudinal septo-temporal axis) and lower it into the radiatum as described previously.</li>
		<li>Place a second LFP electrode into a CA1 site (200 - 800 &#181;m septal or temporal from the first LFP) and observe that CA1 theta oscillations can synchronize over relatively large distances.</li>
	</ol>
	</li>
	<li>Theta oscillations across layers
	<ol>
		<li>To test the properties of theta oscillations across hippocampal layers, leave a reference LFP electrode in a CA1 radiatum site. Starting from just above the stratum oriens, lower a second electrode into the pyramidal cell layer and through the radiatum. Observe a gradual inversion of the LFP signal (relative phase reversal) across the pyramidal layer. 
		NOTE: For representative examples of these types of activity, see Figure 2B. Examples of laminar/depth profiles and Current Source Density (CSD) analysis illustrating the site of phase reversal in isolated hippocampi have been published previously.</li>
	</ol>
	</li>
</ol>
Table 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">SUCROSE SOLUTION (1X) FOR ISOLATED HIPPOCAMPUS</td>
		</tr>
		<tr>
			<td colspan="2">(Stock solution)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound</td>
			<td>MW</td>
			<td>Final Conc. (mM)</td>
			<td>Amount for 1 L (g)</td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>342.3</td>
			<td>252</td>
			<td>86.26 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.01</td>
			<td>24</td>
			<td>2.020 g</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>180.2</td>
			<td>10</td>
			<td>1.800 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.55</td>
			<td>3</td>
			<td>0.223 g</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>120.4</td>
			<td>2</td>
			<td>0.241 g</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>120</td>
			<td>1.25</td>
			<td>0.150 g</td>
		</tr>
		<tr>
			<td>CaCl2.2H2O [1 M] stock</td>
			<td>147</td>
			<td>1.2</td>
			<td>120 &#956;L / 0.1 L *</td>
		</tr>
		<tr>
			<td colspan="4">* add 360 &#956;L CaCl2 [1 M] for 0.3 L oxygenated sucrose solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">pH = 7.4 when oxygenated, Osm 310 - 320</td>
		</tr>
	</tbody>
</table>
Table 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">STANDARD ACSF SOLUTION (5X) FOR PERFUSION</td>
		</tr>
		<tr>
			<td colspan="2">(Stock solution)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound</td>
			<td>MW</td>
			<td>Final Conc. (mM)</td>
			<td>Amount for 2 L&#160; 5X</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.44</td>
			<td>126</td>
			<td>73.6</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.01</td>
			<td>24</td>
			<td>20.2</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>180.2</td>
			<td>10</td>
			<td>18</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.55</td>
			<td>&#9830; 4.5</td>
			<td>3.355</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>120.4</td>
			<td>2</td>
			<td>2.41</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>120</td>
			<td>1.25</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>Ascorbate</td>
			<td>176.1</td>
			<td>0.4</td>
			<td>0.705</td>
		</tr>
		<tr>
			<td>CaCl2.2H2O [1 M] stock</td>
			<td>147</td>
			<td>2</td>
			<td>2 mL / L *</td>
		</tr>
		<tr>
			<td colspan="4">* add 2 mL CaCl2 [1 M] for 1 L aCSF (1x) oxygenated solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">pH = 7.4 when oxygenated, Osm 310 - 320</td>
		</tr>
		<tr>
			<td colspan="4">&#9830; A slightly elevated [K+]o is used for this aCSF solution (compared to normal aCSF 2.5 mM KCl) to increase the excitability of hippocampal networks and facilitate the emergence of theta oscillations.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 - sodium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-25</td>
			<td>brain extraction</td>
		</tr>
		<tr>
			<td>Scalpel Handle #4, 14cm</td>
			<td>WPI</td>
			<td>500237</td>
			<td>brain extraction</td>
		</tr>
		<tr>
			<td>Filter forceps, flat jaws, straight (11cm)</td>
			<td>WPI</td>
			<td>500456</td>
			<td>brain extraction</td>
		</tr>
		<tr>
			<td>Paragon Stainless Steel Scalpel Blades #20</td>
			<td>Ultident</td>
			<td>02-90010-20</td>
			<td>brain extraction</td>
		</tr>
		<tr>
			<td>Fine Point Curved Dissecting Scissors</td>
			<td>Thermo Fisher Scientific</td>
			<td>711999</td>
			<td>brain extraction</td>
		</tr>
		<tr>
			<td>Teflon (PTFE) -coated thin spatula</td>
			<td>VWR</td>
			<td>82027-534</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Hayman Style Microspatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-25A</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Lab spoon</td>
			<td>Fisher Scientific</td>
			<td>14-375-20</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Droper</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Razor blades Single edged</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Lens paper (4X6 inch)</td>
			<td>VWR</td>
			<td>52846-001</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Glass petri dishes (100 x 20 mm)</td>
			<td>VWR</td>
			<td>25354-080</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Plastic tray for ice; size 30 x 20 x 5 cm</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr>
			<td>Single Inline Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td>perfusion system</td>
		</tr>
		<tr>
			<td>Aquarium air stones for bubbling</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr>
			<td>Tygon E-3603 tubing (ID 1/16 OD 1/8)</td>
			<td>Fisherbrand</td>
			<td>14-171-129</td>
			<td>perfusion system</td>
		</tr>
		<tr>
			<td>Electric Skillet</td>
			<td>Black &#38; Decker</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr>
			<td>95% O2/5% CO2 gas mixture (carbogen)</td>
			<td>Vitalaire</td>
			<td>SG466204A</td>
			<td>perfusion system</td>
		</tr>
		<tr>
			<td>Glass bottles/flasks (4 x 1 L)</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr>
			<td>Submerged recording Chamber</td>
			<td>custom design (FM)</td>
			<td>n.a.</td>
			<td>Commercial alternative may be used</td>
		</tr>
		<tr>
			<td>Glass pipettes (1.5 / 0.84 OD/ID (mm) )</td>
			<td>WPI</td>
			<td>1B150F-4</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>Hum Bug 50/60 Hz Noise Eliminator</td>
			<td>Quest Scientific</td>
			<td>Q-Humbug</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular devices</td>
			<td>MULTICLAMP</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>Multiclamp 700B Commander Program</td>
			<td>Molecular devices</td>
			<td>MULTICLAMP</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>Digital/Analogue converter</td>
			<td>Molecular devices</td>
			<td>DDI440</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>PCLAMP10</td>
			<td>Molecular devices</td>
			<td>PCLAMP10</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Newport</td>
			<td>n.a.</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>Micromanipulators (manually operated )</td>
			<td>Siskiyou</td>
			<td>MX130</td>
			<td>electrophysiology (LFP)</td>
		</tr>
		<tr>
			<td>Micromanipulators (automated)</td>
			<td>Siskiyou</td>
			<td>MC1000e</td>
			<td>electrophysiology (patch)</td>
		</tr>
		<tr>
			<td>Audio monitor</td>
			<td>A-M Systems</td>
			<td>Model 3300</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>Micropipette/Patch pipette puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td>electrophysiology</td>
		</tr>
		<tr>
			<td>Custom-built upright fluorescence microscope</td>
			<td>Siskiyou</td>
			<td>n.a.</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Analogue video camera</td>
			<td>COHU</td>
			<td>4912-2000/0000</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Digital frame grabber with imaging software</td>
			<td>EPIX, Inc</td>
			<td>PIXCI-SV7</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Olympus 2.5x objective</td>
			<td>Olympus</td>
			<td>MPLFLN</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Olympus 40x water immersion objective</td>
			<td>Olympus</td>
			<td>UIS2 LUMPLFLN</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Custom-made light-emitting diode (LED) system</td>
			<td>custom</td>
			<td>n.a.</td>
			<td>optogenetic stimulation (Amhilon et al., 2015)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>PVChY mice</td>
			<td>In house breeding</td>
			<td>n.a.</td>
			<td>Offspring obtained from cross-breeding the PV-Cre line with Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP</td>
		</tr>
	</tbody>
</table>

in vitro recording of theta oscillations in the mouse hippocampus

Source: Manseau, F., et.al. <a href="https://app.jove.com/v/55851">Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit.</a> J. Vis. Exp. (2017).
This video demonstrates the recording of rhythmic neuronal network activity, also known as theta oscillations, in an isolated hippocampus. Electrodes are inserted into various layers and spatial positions to detect and analyze theta oscillations. This shows the unique neuronal compositions and the connectivity of the neuronal network across different layers of the hippocampus.

<img alt="Figure 1" src="/files/ftp_upload/22950/22950_Figure1.jpg" /> 
Figure 1: Dissection Procedure for the Isolated Intact Hippocampus Preparation.
(a) General view of the dissection setup. Top right: carbogenated ice-cold sucrose solution flask (1); bottom left: ice-filled plastic tray (2) holding the dissection dish covered with lens paper (3); the cold holding chamber containing sucrose solution (4); and a set of surgical tools (5). (b) View of the mouse brain before hemisection on the dissection dish. (c) Recovery of the hemisected brain in the cold holding chamber and zoomed view (inset) of the left brain hemisphere before inserting the small end of the coated spatula under the septum. (d) A coated spatula placed under the isolated hippocampus, along the CA1/SUB region, with the remaining brain tissue is pulled out from underneath.
<img alt="Figure 2" src="/files/ftp_upload/22950/22950_Figure2.jpg" /> 
Figure 2: Configuration of the Setup for Recording in vitro Theta Oscillations from the Intact Hippocampal Preparation in the Submerged Recording Chamber.
(a) The isolated hippocampus is shown with a layout of hippocampal regions and multiple electrodes placed in four different recording sites distributed septotemporally (indicated by white asterisks). In the view of the recording chamber platform shown above (inset i), the inlet and outlet for fast-perfusion flow are indicated by numbers (1, 2). In the enlarged image of the hippocampus shown below (inset ii), a single electrode is placed in the midsepetotemporal CA1, and fibers of the alveus are readily visible, running diagonally toward the subiculum. S: septal, T: temporal, f/fx: fimbria-fornix. (b) Schematic representation of the organization of CA1 layers with representative LFP traces recorded simultaneously from stratum oriens (grey) and stratum radiatum (black). Note the inverted phase of signals between the two layers. Alv: stratum alveus, PR: stratum pyramidale, SR: stratum radiatum. SLM: Stratum Lacunosum Moleculare. (c) Example LFP trace showing spontaneous theta oscillation recorded from the CA1/SUB area (20-sec segment) and 2-sec expanded segments (below) from the unfiltered signal; band-pass filtered for theta frequencies (0.5 - 12 Hz); slow gamma (25 - 55 Hz); and fast gamma (125 - 250 Hz).

In Vitro Recording of Theta Oscillations in the Mouse Hippocampus

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

Begin with an isolated mouse hippocampus in a recording setup.
Lower the local field potential or LFP electrode to the surface of the hippocampus. This electrode can detect extracellular voltage changes indicative of neuronal network activity.
Insert the electrode and observe the voltage changes.
Insert further into the lower layer and observe the rhythmic neuronal network activity, known as theta oscillations.
Position a second electrode at a distance from the first within the same layer to record theta oscillations across a broader region.
Monitor the synchronization of theta oscillations over considerable distances. This shows the connectivity of the neuronal network.
To record theta oscillations through different hippocampal layers, begin by placing the second electrode in the upper layer.
Gradually lower the electrode through different layers.
Record and observe the inversion of the oscillation pattern, which reflects the unique neuronal composition and function of each layer of the hippocampus.

in-vitro-recording-of-theta-oscillations-in-the-mouse-hippocampus

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

33 visualizações. Fonte: Manseau, F., et.al. Sintonia na Banda Theta do Hipocampo In Vitro: Metodologias para Gravação do Circuito Septo-Hipocampal de Roedores Isolados. J. Vis. Exp. (2017). Este vídeo demonstra a gravação da atividade da rede neuronal rítmica, também conhecida como oscilações, em um hipocampo isolado. Os eletrodos são inseridos em várias camadas e posições espaciais para detectar e analisar as oscilações. Isso mostra as composições neuronais únicas e a conectividade da rede ne...

Vídeo: Registro in vitro de oscilações no hipocampo de camundongos - Conceito

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.

Gravação espacialmente restrita oscilações no hipocampo de ratos de comportar-se

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

JoVE Journal

Neurociência

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.

In Vitro Recording of Theta Oscillations in the Mouse Hippocampus

Transcrição

In Vitro Recording of Theta Oscillations in the Mouse Hippocampus