Name: tuning in the hippocampal theta band in vitro: methodologies for recording from the isolated rodent septohippocampal circuit
Uploaded: 2017-08-02T13:00:00
Description: Watch this Scientific Journal Video about Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit at JoVE.com

This work was supported by the Canadian Institutes of Health Research and Natural Sciences.

All procedures have been performed according to protocols and guidelines approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care.
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 ...

<ol>
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While electrophysiological recordings from acute hippocampal slices constitute a standard in vitro technique, the methods presented here differ substantially from the classic approach. Unlike the thin slice preparations where specific cell layers are visible at the surface and can be examined directly, the intact hippocampal preparations are more akin to in vivo configurations where electrodes are lowered into targeted brain regions while crossing through individual layers. The integrity of the hippocam...

Hippocampal theta oscillations (4 - 12 Hz) are amongst the most predominant forms of rhythmic activity in the mammalian brain and are believed to play key roles in cognitive functions such as processing of spatiotemporal information and formation of episodic memories1,2,3. While several in vivo studies that highlight the relationship of theta-modulated place-cells with spatial navigation and lesion studies, as well as clinical evidence, support the view that hippocampal theta oscillations are involved in memory formation4,5,6, the mechanisms associated with generation of hippocampal theta oscillations are still not fully understood. Early in vivo investigations suggested that theta activity depended mainly on extrinsic oscillators, in particular rhythmic input from afferent brain structures such as the septum and entorhinal cortex7,8,9,10. A role for intrinsic factors - internal connectivity of hippocampal neural networks together with the properties of hippocampal neurons - was also postulated based on in vitro observations11,12,13,14,15,16,17,18. However, apart from a few landmark studies19,20,21, difficulties in developing approaches that could replicate physiologically realistic population activities in simple in vitro slice preparations have, for a long time, delayed more detailed experimental examination of the intrinsic abilities of the hippocampus and related areas to self-generate&#160;theta oscillations.
An important downside of the standard in vitro thin-slice experimental setting is that the 3D cellular and synaptic organization of brain structures is usually compromised. This means that many forms of concerted network activities based on spatially distributed cell assemblies, ranging from localized groups (&#8804;1 mm radius) to populations of neurons spread across one or more brain areas (&#62;1 mm), cannot be supported. Given these considerations, a different type of approach was needed to study how theta oscillations emerge in the hippocampus and propagate to related cortical and subcortical output&#160;structures.
In recent years, the initial development of the &#34;complete septo-hippocampal&#34; preparation to examine bidirectional interactions of the two structures22, and the ensuing evolution of the &#34;isolated hippocampus&#34; preparation, have revealed that intrinsic theta oscillations occur spontaneously in the hippocampus lacking external rhythmic input23. The value of these approaches lies on the initial insight that the whole functional structure of these regions had to be preserved in order to function as a theta rhythm generator in vitro22.

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			<td height="21" style="height:21px;width:319px;">Reagents</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4 - sodium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631-25G</td>
			<td></td>
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		<tr height="30">
			<td height="30" style="height:30px;width:319px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
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			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Standard Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-25</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #4, 14cm</td>
			<td>WPI</td>
			<td>500237</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter forceps, flat jaws, straight (11cm)</td>
			<td>WPI</td>
			<td>500456</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paragon Stainless Steel Scalpel Blades #20</td>
			<td>Ultident</td>
			<td>02-90010-20</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Point Curved Dissecting Scissors</td>
			<td>Thermo Fisher Scientific</td>
			<td>711999</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teflon (PTFE) -coated thin spatula</td>
			<td>VWR</td>
			<td>82027-534</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hayman Style Microspatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-25A</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab spoon</td>
			<td>Fisher Scientific</td>
			<td>14-375-20</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Droper</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades Single edged</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lens paper (4X6 inch)</td>
			<td>VWR</td>
			<td>52846-001</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass petri dishes (100 x 20 mm)</td>
			<td>VWR</td>
			<td>25354-080</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Single Inline Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aquarium air stones for bubbling</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Electric Skillet</td>
			<td>Black &#38; Decker</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">95% O2/5% CO2 gas mixture (carbogen)&#160;</td>
			<td>Vitalaire</td>
			<td>SG466204A</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottles/flasks (4 x 1 L)</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Submerged recording Chamber</td>
			<td>custom design (FM)</td>
			<td>n.a.</td>
			<td style="width:247px;">Commercial alternative may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass pipettes (1.5 / 0.84 OD/ID (mm) )</td>
			<td>WPI</td>
			<td>1B150F-4</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hum Bug 50/60 Hz Noise Eliminator</td>
			<td>Quest Scientific</td>
			<td>Q-Humbug</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular devices</td>
			<td>MULTICLAMP</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B Commander Program</td>
			<td>Molecular devices</td>
			<td>MULTICLAMP</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital/Analogue converter</td>
			<td>Molecular devices</td>
			<td>DDI440</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCLAMP10</td>
			<td>Molecular devices</td>
			<td>PCLAMP10</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibration isolation table&#160;</td>
			<td>Newport</td>
			<td>n.a.</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulators (manually operated )</td>
			<td>Siskiyou&#160;</td>
			<td>MX130</td>
			<td>electrophysiology (LFP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulators (automated)</td>
			<td>Siskiyou&#160;</td>
			<td>MC1000e</td>
			<td>electrophysiology (patch)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Audio monitor&#160;</td>
			<td>A-M Systems</td>
			<td>Model 3300</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette/Patch pipette puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom-built upright fluorescence microscope</td>
			<td>Siskiyou</td>
			<td>n.a.</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Analogue video camera</td>
			<td>COHU</td>
			<td>4912-2000/0000</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital frame grabber with imaging software</td>
			<td>EPIX, Inc</td>
			<td>PIXCI-SV7</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus 2.5x objective</td>
			<td>Olympus</td>
			<td>MPLFLN</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus 40x water immersion objective</td>
			<td>Olympus</td>
			<td>UIS2 LUMPLFLN</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom-made light-emitting diode (LED) system&#160;</td>
			<td>custom</td>
			<td>n.a.</td>
			<td>optogenetic stimulation (Amhilon et al., 2015)</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:319px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">PV::Cre (KI) mice</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock number 008069</td>
			<td style="width:247px;">Allow&#160; Cre-directed gene expression in PV interneurons</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Constitutive-conditional Ai9 mice (R26-lox-stop-lox-tdTomato (KI))</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock number 007905</td>
			<td style="width:247px;">Express TdTomato following Cre-mediated recombination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock 
			number 012569</td>
			<td style="width:247px;">Express the improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVChY mice</td>
			<td>In house breeding</td>
			<td>n.a.</td>
			<td style="width:247px;">Offspring obtained from cross-breeding the PV-Cre line with Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP</td>
		</tr>
	</tbody>
</table>

tuning in the hippocampal theta band in vitro: methodologies for recording from the isolated rodent septohippocampal circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

This section illustrates examples of results that can be obtained by studying theta oscillations in the mouse isolated hippocampal preparation in vitro. The dissection procedure for extracting the isolated hippocampus is illustrated in Figure 1. Using this preparation, intrinsic theta oscillations can be examined during placement of multiple field electrodes, recording overall activity and synchronized synaptic inputs to neuronal populations in diffe...

Watch this Scientific Journal Video about Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit at JoVE.com

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

The overall goal of this protocol is to present procedures for extracting the whole hippocampal preparation and exploring the generation of rhythmic neuronal network using field, unitary and patch-clamp recordings as well as optogenetic stimulation. This method can help answer key questions in the neuroscience field of learning and memory by greatly facilitating the study of cellular and synaptic mechanisms that underlie rhythmic oscillations in the hippocampus. The main advantage of this technique is that it uses an optimized preparation to investigate the circuits in detailed oscillations, which play a crucial role in hippocampal dependent memory information.To begin this procedure, place the brain onto the dissection dish in an upright position, remove the cerebellum with a razor blade, then cut the brain in half along the mid sagittal plane and return the two isolated hemispheres to the holding chamber. Next, place the single hemisected brain upright onto the dissection dish. Rotate the dish until the mid sagittal structures are facing the experimenter and the embedded outline of the septal complex is visible as a thin pear shaped layer of tissue interior to the thalamus.Then, insert a coated spatula beneath the septum and move the tip down until the dissection dish is reached, sever the fibers that connect the septal area cordially. Now, repeat the same operation along the anterior edge of the septum and cut the fibers that connect to the frontal part of the brain. With the spatula lightly holding the inner part of the cortex just above the hippocampus in an upright position, use the microspatula to carefully pull down the thalamic, hypothalamic and the remaining brain stem nuclei.Subsequently, use the spatula to cut off and remove the pulled away tissue. Afterward, insert the coated spatula in the lateral ventricle underneath the rostral end of the dorsal hippocampus. Hold the spatula horizontally, aligned with the mid-sagittal plane of the hemisected brain and slide it through the smooth contour of the ventricular walls, until the tip emerges cordially.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, then, apply the microspatula on the external side of this layer and press it against the coated spatula to slice through the connecting fibers. To complete the extraction, rotate the dissection dish and insert the coated spatula beneath the ventral hippocampus. Lightly hold the hippocampus with the spatula and cut through the endocrinal connections using a slicing motion against the spatula.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 to the fornix. After that, transfer the preparation to room temperature sucrose solution and let it recover for 15 to 30 minutes before transferring to the recording chamber.In this step, set up the gravity fed perfusion system to enable continuous high speed in flow of oxygenated ACSF. Next, stop the 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.Place the preparation in the center of the recording chamber, with the smooth surface of CA1 and subiculum on top. Stabilize the hippocampus with small weights of the septal and temporal extremities and restart the ACSF flow. In this procedure, lower the LFP electrode to the surface of the hippocampus.Advance the LFP electrode through the parameter layer and observe the increase in extracellular spiking activity as single unit discharge from individual neurons is detected. Lower the electrode further and note that spiking begins to fade again as the tip crosses into the radiatom. Observe that a clearly visible network oscillation in the theta frequency range, becomes apparent and reaches maximum amplitude as the recording location is lowered through the radiatom.To test the spatial properties of spontaneous theta oscillations across the CA1 region, place a second LFP electrode into a CA1 site and observe that CA1 theta oscillations synchronize over large distances. To test the properties of theta oscillations across hippocampal layers, leave a reference LFP electrode in a CA1 radiatom site. Starting from just above the stratum oriens, lower a second electrode into the parameter cell layer and through the radiatom.Observe a gradual inversion of the LFP signal across the parameter layer. To test gamma oscillations and theta gamma coupling in the intact hippocampus, place a field electrode at the CA1 subiculum border and lower it until it sits at the interface between the parameter and molecular layers. At this level, a field potential displaying clear gamma oscillations with changes in amplitude that phase locked to the local theta rhythm can be recorded.Then, adjust the scaling to observe the slow time scale of theta gamma coupling. Note that gamma bursts occur in two distinct frequency bands which can be revealed by band during the ongoing LFP signal, in the slow and fast gamma range. For whole cell patch clamp recording during in vitro hippocampal theta oscillations, use fluorescence video microscopy with low and high power magnification to visualize tdTomato positive interneurons located near the surface of a hippocampal preparation from a PV tom mouse.Under low magnification view of the hippocampus, place an LFP electrode in CA1 subiculum to monitor theta oscillations while preparing for patch clamp experiments. Then, switch to 40x magnification and immerse the objective over the target region. Lower it until the top layers become visible, under the fluorescence microscopy, select the fluorescent PV tom cell and approach with a patch pipette filled with standard intercellular solution.Once in whole cell configuration, examine the physiological properties of the identified PV cell during spontaneous hippocampal oscillations. Observe the intecellular membrane potential recording from PV cells that are characterized from fast spiking behavior and bursts of action potentials synchronized to the ongoing CA1 subiculum theta rhythm. In this procedure, place an LFP electrode in the CA1 subiculum area and patch a nearby parameter cell in the isolated hippocampus of a mouse, expressing the blue light sensitive excitatory opcin, CHR2 in PV interneurons.Place an optic fiber light guide above the hippocampal preparation and center it on the recorded region. Use blue light from a LED source for optic genetic stimulation, which consists of 10 to 20 millisecond light pulses or sin wave voltage commands delivered at theta frequencies. In current clamp, characterize the activity of the recorded cell during spontaneous theta oscillations.Then, start the stimulation protocol and record the light responses. Observe that field oscillations and synaptic activity and the recorded neuron, become increasingly synchronized during optogenetic stimulation and that rhythmic pacing of PV cells results in a robust control of both frequency and power of theta oscillations. Shown here, is the electrophysiological activity recorded from a parameter cell during theta oscillations.Current clamp traces shows spontaneous but not rhythmic firing at rest and inhibitory post synaptic potentials that were not clearly synchronized with the slowly emerging LFP oscillation. Voltage clamp recordings show that the corresponding inhibitory post synaptic currents have the reversal potential at around minus 70 millivolts. And here, is the electrophysiological activity recorded from a fast spiking fluorescent PV interneuron during theta oscillations.In current clamp, this cell was spontaneously firing at rest and was strongly driven by rhythmic excitatory post synaptic potentials that were phased locked with the stable LFP oscillation. In voltage clamp recordings, the excitatory post synaptic current reversal potential was approximately at zero millivolts. Once mastered, this technique can be performed in two to three hours, while attempting this procedure, it's important to remember to oxygenate a solution vigorously and use a perfusion system allowing high speed but steady flow of armed ACSF over the preparation during the electrophysiological recordings.Following this procedure, other methods such as optogenetics stimulation or silencing of specific cell type populations, can be used in order to answer additional questions such as identifying the cellular subtypes that form theta oscillators in the hippocampus. After its development, this technique paved a way for researchers in the field of neuroscience to systematically explore the dynamics of theta oscillations across the septal temporal axis of the hippocampus in vitro. After watching this video, you should have a good understanding of how to extract a whole hippocampus preparation in order to explore the function of rhythmic neuronal networks using field, unit and patch clamp recordings, as well as optogenetic stimulation.

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