Name: investigating long-term synaptic plasticity in interlamellar hippocampus ca1 by electrophysiological field recording
Uploaded: 2019-08-11T14:00:00
Description: Watch this Scientific Journal Video about Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording at JoVE.com

This work was supported by Incheon National University (International Cooperative) Research Grant. We will like to thank Ms. Gona Choi for assisting with some data collection.

All animals were treated in accordance with the guidelines and regulations from the Animal Care and Use of Laboratory of National Institute of Health. All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of City University of Hong Kong and Incheon National University.
1. In vivo field recording
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	<li>Animal preparation
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		<li>Inject urethane (0.06 g per 25 g weight) intraperitoneally to anesthetize mouse. Supplement with intramuscu...

<ol>
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The protocol demonstrates the method to induce long-term synaptic plasticity in vivo as well as from brain slices in the longitudinal CA1-CA1 axis of the hippocampus in vitro. The steps outlined give enough details for an experimenter to investigate LTP and LTD in a longitudinal hippocampal CA1-CA1 connection. Practice is needed to hone the skills required to successfully record field excitatory potentials.
In addition to needing practice, there are several critical steps that are essential to...

The hippocampus has been widely used in cognitive studies1,2,3. The hippocampal lamellar network in the transverse axis forms the tri-synaptic circuitry that is made up of the dentate gyrus, CA3, and CA1 regions. The lamellar network is considered to be a parallel and independent unit4,5. This lamellar viewpoint has influenced the use of transverse orientation and transverse slices for both in vivo and in vitro electrophysiological studies of the hippocampus. In light of emerging research, the lamellar hypothesis is being reevaluated6 and attention is also being given to the interlamellar network of the hippocampus. With regards to the hippocampal interlamellar network, the CA3 region has long been investigated7,8,9,10, however the longitudinal CA1 hippocampal region has received relatively little attention until recently. With regards to the CA1 interlamellar network, the short-term synaptic properties along the dorsoventral longitudinal hippocampal CA1 axis of rats have been shown to vary11. Also, clusters of hippocampal cells responding to the phase and the place were found to be arranged systematically along the longitudinal axis of the hippocampus in rats, undergoing a short term memory task12. Also, epileptic seizure activities were found to be synchronized along the whole hippocampus along the longitudinal axis13.
Most studies of the longitudinal CA1 hippocampal region however, have utilized input from the CA3 to the CA1 regions11,14,15. Using a unique protocol to make longitudinal brain slices, our previous work demonstrated the associational connectivity of CA1 pyramidal neurons along the longitudinal axis and implicated its ability to process neuronal signaling effectively16. However, there is a need to determine whether the CA1 pyramidal neurons along the longitudinal axis without transverse input can support long term synaptic plasticity. This finding can add another angle into investigations of neurological issues pertaining to the hippocampus.
The ability of neurons to adapt the efficacy of information transfer is known as synaptic plasticity. Synaptic plasticity is implicated as the underlying mechanism for cognitive processes such as learning and memory17,18,19,20. Long-term synaptic plasticity is demonstrated as either long-term potentiation (LTP), which represents the strengthening of neuronal response, or long-term depression (LTD), which represents the weakening of neuronal response. Long-term synaptic plasticity has been studied in the transverse axis of the hippocampus. However, this is the first study to demonstrate long-term synaptic plasticity in the hippocampal longitudinal axis of CA1 pyramidal neurons.
Building from a protocol used by Yang et al.16, we designed the protocol to demonstrate LTP and LTD in the hippocampal longitudinal axis of CA1 pyramidal neurons. We used C57BL6 male mice with ages ranging between 5-9 weeks old for in vitro experiments and 6-12 weeks old for in vivo experiments. This detailed article shows how longitudinal hippocampal brain slices from mice were obtained for in vitro recordings and how in vivo recordings were recorded in the longitudinal axis. For in vitro recordings, we investigated directional specificity of longitudinal CA1 synaptic plasticity by targeting the septal and temporal end of the hippocampus. We also investigated layer specificity of the longitudinal CA1 synaptic plasticity by recording from the stratum oriens and stratum radiatum of the hippocampus. For in vivo recordings, we investigated the angles that best correspond to the longitudinal direction of the hippocampus.
Using both in vivo and in vitro extracellular field recordings, we observed that the longitudinally connected CA1 pyramidal neurons presented with LTP, not LTD. The transverse orientation involving both CA3 and CA1 neurons, however, supports both LTP and LTD. The distinction in the synaptic capabilities between the transverse and the longitudinal orientation of the hippocampus could speculatively signify differences in their functional connectivity. Further experiments are needed to decipher the differences in their synaptic capabilities.

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			<td height="20" style="height:20px;width:163px;">Atropine Sulphate salt monohydrate, &#8805;97% (TLC), crystalline</td>
			<td style="width:63px;">Sigma-Aldrich</td>
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			<td style="width:63px;">Stored in Dessicator</td>
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			<td height="20" style="height:20px;width:163px;">Axon Digidata 1550B</td>
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			<td height="20" style="height:20px;">Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
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			<td height="20" style="height:20px;width:163px;">Clampex 10.7</td>
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			<td>50-99-7</td>
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			<td height="21" style="height:21px;width:163px;">Isoflurane</td>
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			<td height="41" style="height:41px;width:163px;">Matrix electrodes, Tungsten</td>
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			<td>Sigma-Aldrich</td>
			<td>7778-77-0</td>
			<td>Stored in Dessicator</td>
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			<td height="21" style="height:21px;width:163px;">Pump</td>
			<td>Longer precision pump Co., Ltd</td>
			<td>T-S113&#38;JY10-14</td>
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			<td>Sigma-Aldrich</td>
			<td>63148-62-9</td>
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			<td height="40" style="height:40px;width:163px;">Sodium Bicarbonate, BioXtra, 99.5-100.5%</td>
			<td>Sigma-Aldrich</td>
			<td style="width:100px;">144-55-8</td>
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			<td height="40" style="height:40px;width:163px;">Sodium Chloride, BioXtra, &#8805;99.5% (AT)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:100px;">7647-14-5</td>
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			<td height="40" style="height:40px;width:163px;">Sodium phosphate monobasic, powder</td>
			<td>Sigma-Aldrich</td>
			<td style="width:100px;">7558-80-7</td>
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			<td height="20" style="height:20px;width:163px;">Sucrose, &#8805; 99.5% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:100px;">57-50-1</td>
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			<td height="20" style="height:20px;width:163px;">Temperature controller</td>
			<td>Warner Instruments</td>
			<td style="width:100px;">TC-324C</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Tungsten microelectrodes</td>
			<td>FHC</td>
			<td align="right">20843</td>
			<td></td>
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			<td height="20" style="height:20px;width:163px;">Urethane, &#8805;99%</td>
			<td>Sigma-Aldrich</td>
			<td style="width:100px;">51-79-6</td>
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			<td>Grant Instruments</td>
			<td>SAP12</td>
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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 explored long-term synaptic plasticity of longitudinal CA1 pyramidal neurons of the hippocampus using extracellular field recordings both in vivo and in vitro. LTP and LTD are facets of long-term synaptic plasticity that have been demonstrated in the transverse axis of the hippocampus to be unidirectional.
We showed here that using longitudinal hippocampal brain slices, there is LTP in the CA1 longitudinal axis of the hippocampus. We prepared longitudinal slices of the hippocampus along the...

Watch this Scientific Journal Video about Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording at JoVE.com

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

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.

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 ...

Long-term synaptic plasticity, specifically, long-term potentiation and long-term depression have been investigated widely along the transverse orientation of the hippocampus. It has also been looked at along the longitudinal orientation. However, when it comes to the longitudinal orientation, very little attention has been given to the longitudinal CA1 axis of the hippocampus with respect to synaptic plasticity.A previous publication from our lab have shown that the CA1 pyramidal neurons of the hippocampus form associational connections that could support synaptic plasticity. For further understanding of this protocol, it is recommended that you refer to the previous publication with the given reference below. Inject urethane to anesthetize the mouse.Place the fully anesthetized mouse on a heating pad set to 37 degrees Celsius. Apply eye gel to moisten the eyes. Fix the skull of the mouse firmly using the eye clamp.The eye clamp gives the freedom of mobility for the experimenter during surgical procedures. And it can be particularly useful for experiments involving the auditory cortex, as well. However, much care must be taken to get the exact stereotaxic coordinates.Separate the subcutaneous tissue and muscles at the end of the interparietal and occipital bone of the mouse with the scalpel. Blot the dura matter dry with cotton swab. Then you puncture the cisterna magna to drain the cerebrospinal fluid.Attach a cotton swab to keep draining the cerebrospinal fluid. Cut and remove the skin on the scalp and keep the exposed region dry. Mark the points corresponding to the hippocampal region using the aid of a veniyar caliper.Make an incision in the skull above the dorsal region of the hippocampus along the marked points using a scalpel or high speed drill while observing under a microscope. Keep the exposed brain tissue moist by applying physiological saline or an inert oil. Fix and position the stimulation and recording electrodes firmly in the stereotaxic holder.Adjust the stimulation and recording electrodes above CA1 dorsal hippocampus. Use the mouse brain in stereotaxic coordinate as a guide in locating the coordinate for dorsal longitudinal CA1 hippocampal region. For recordings using multichannel electrodes, first locate your stereotaxic coordinates for the CA1 hippocampal region using the first channel.Position the remaining electrodes such that the angle of stimulation and recording electrodes are in the range of 30 degrees to 60 degrees in relation to the midline from bregma point. This angle corresponds to the longitudinal orientation of the CA1 hippocampal region. Turn on the recording system.Open the neural software for recording and data acquisition. Lower the recording and stimulation electrodes slowly using the micromanipulator until it just touches the surface of the brain. Observe an increase in the impedance just when the electrode touches the brain surface.This can be observed in the red channel shown on the screen. Slowly lower the electrodes to the approximate depth corresponding to chosen stereotaxic coordinates for the CA1 hippocampus. Give stimulation til a stable evoke-filled excitatory post-synaptic potential is observed.Perform an input-output curve to detect maximum stimulus intensity. Use the input-output curve to set the baseline intensity and to set the stimulus intensity for evoking high frequency stimulation or low frequency stimulation. Record the local full potential for an hour after high frequency stimulation or low frequency stimulation.Export data and analyze using software of choice. Poor 400 ml of already-prepared slicing solution into a separate flask and oxygenate for approximately 20 minutes. Poor 200 ml of the oxygenated slicing solution.Cover in para film and transfer to a 80 degrees freezer for approximately 20 minutes to make a slush. Poor the remaining 200 ml of slicing solution in a brain slice holding chamber and keep in water bot of 32 degrees Celsius with continuous bubbling. Bring out chilled slicing solution and poor approximately 50 ml in a beaker.Decapitate the anesthetized mouse. Cut the skin covering the skull of the mouse and cut the skull piece along the midline to the occipital bone. Open the skull with blunt forceps.Gently scoop out the brain with a spatula and place in the chilled slicing solution in the beaker. Separate the two brain hemispheres along the midline with a scalpel blade. Isolate the hippocampus by gently detaching it from the cortex with a spatula.Cut out the septal and temporal end of the isolated hippocampus. Apply a small amount of glue to the slicing plate. For longitudinal CA1 hippocampal slice, attach the CA3 region of the hippocampus to the slicing plate with the glue.For transverse slice, which will serve as control, attach the ventral end of the hippocampus to the slicing plate of the vibratome. Place it in the slicing chamber. Set the vibratome parameters.Slice the attached hippocampus with the vibratome. A good longitudinal CA1 hippocampal brain slice will have one layer of CA1, and two layers of dentate gyrus. Also, the stratum oriens and the stratum radiatum of the CA1 region will be present.Alternatively, a transverse hippocampal brain slice will have the dentate gyrus CA3 and CA1 regions in tact. Transfer the brain slices to the brain slice holding chamber in the water bot. Incubate for 20 minutes at a temperature of 32 degrees Celsius and bring to room temperature for recovery.Continuously superfuse the ACS serf into the recording chamber at the speed of 2 ml per minute. Transfer the brain slice into the recording chamber, adjust the position of the brain slice with blunt forceps and hold it in place with a hap. Turn on the software for data acquisition.Longitudinal brain slice. For longitudinal slices, position the stimulation and recording electrode in the stratum oriens. Place the stimulation electrode on either the septal or temporal side of the brain slice with recording from the same layer.Alternatively, position the stimulation and recording electrode in the stratum radiatum, place the stimulation electrode in either the septal or temporal side of the brain slice. For transverse slices as control, position the stimulation electrode on the CA3 Schaffer collateral pathway region, and the recording electrode on the CA1 region. Turn on the isolator stimulus generator and give stimulation.Adjust the recording electrode depth til a stable evoke excitatory post-synaptic-filled potential is observed. Perform an input-output curve to detect maximum stimulus intensity. Use the input-output curve to set the stimulus intensity for baseline recording evoking high frequency stimulation or low frequency stimulation.Refer to the attached PDF for parameters in inducing lone-term potentiation or long-term depression. Evoked excitatory post-synaptic potential increased in response to high frequency stimulation in vivo. This shows that longitudinal hippocampal CA1 supports long-term potentiation.For longitudinal hippocampal CA1 brain slices, increase in response to high frequency stimulation was observed at both septal and temporal side of stratum oriens and stratum radiatum. This shows that the synaptic plasticity induced along the longitudinal hippocampal CA1 region is not linear or direction specific. Using already established protocols for long-term depression, no long-term depression was induced both in vivo and in vitro.As a conclusion, we have shown you how to investigate long-term synaptic plasticity in the longitudinal hippocampal CA1 region both in vivo and in vitro. More details of the protocol can be found in the PDF accompanying this video to assist you in investigating long-term synaptic plasticity along the longitudinal hippocampal CA1 region in your own labs.

Research

JoVE Journal

Neuroscience

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Forebrain Electrophysiological Recording in Larval Zebrafish

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Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

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Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

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 ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

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 ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...