Name: investigating long-term synaptic plasticity in interlamellar hippocampus ca1 by electrophysiological field recording
Uploaded: 2019-08-11T14:00:00.000+00:00
Duration: 14 min 27 s
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.

<ol>
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We have nothing to disclose.

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 obtaining good results. First, it was shown previously that the angle to which the brain slices were made could either truncate or preserve the longitudinal projections of the pyramidal neurons in the CA1-CA1 region of the hippocampus16. The longitudinal pyramidal neurons project from the transverse neurons at an angle that is nearly perpendicular. As the CA1 neurons propagate in diverse angles within the hippocampus, the longitudinal connection between them lays out along the dorsoventral axis of the hippocampus. Thus, for in vitro recording, the experimenter must keep this in mind to accurately target the CA1-CA1 hippocampal neurons along the longitudinal axis by cutting the isolated hippocampus tissue along the dorsal-ventral axis. Also, for in vivo recordings, the angle at which the stimulation and the recording electrodes are positioned determines whether the results obtained are representative of the longitudinal axis or a mixture of both the transverse and longitudinal axis. Further investigations utilizing CRISPR-Cas9 can be done to confirm whether the evoked response is solely from the CA1 region since it could be a mixture of responses from both the CA1 and the CA3 regions.
Secondly, for in vitro experiments, the experimenter must ensure that the brain slicing solution, ACSF, work bench and all equipment or instruments that come in contact with the brain slice are free from contaminants. Any form of contamination will lead to the deterioration of the integrity or death of the brain slice. Maintaining a clean electrode surface will ensure good and stable recordings for both in vitro and in vivo experiments.
We have shown that the longitudinal hippocampal CA1 network exhibits NMDAR-dependent LTPs, but not LTDs. The trisynaptic circuit, however, presents with both LTP and LTD22,23. This implies that the longitudinal CA1 network and the tri-synaptic circuitry have unique features. Our protocol makes use of only electrophysiological recordings and therefore is limited in finding the difference between these two networks.
The search for a cure for brain diseases such as schizophrenia continues. Decline or deformity of CA1 hippocampal subregions have been linked with some schizophrenic symptoms24,25. The application of our protocol, though basic, has brought to light a unique synaptic capability of the CA1 longitudinal hippocampal subregion. This knowledge is useful in designing experiments that can further investigate this debilitating brain disease along the longitudinal CA1 axis of the hippocampus.

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.

<table><tbody><tr><td>Atropine Sulphate salt monohydrate, &amp;ge;97% (TLC), crystalline</td><td>Sigma-Aldrich</td><td>5908-99-6</td><td>Stored in Dessicator</td></tr><tr><td>Axon Digidata 1550B</td><td></td><td></td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>10035-04-8</td><td></td></tr><tr><td>Clampex 10.7</td><td></td><td></td><td></td></tr><tr><td>D-(+)-Glucose &amp;ge; 99.5% (GC)</td><td>Sigma-Aldrich</td><td>50-99-7</td><td></td></tr><tr><td>Eyegel</td><td>Dechra</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>RWD Life Sciences</td><td>R510-22</td><td></td></tr><tr><td>Magnesium chloride hexahydrate, BioXtra, &amp;ge;99.0%</td><td>Sigma-Aldrich</td><td>7791-18-6</td><td></td></tr><tr><td>Matrix electrodes, Tungsten</td><td>FHC</td><td>18305</td><td></td></tr><tr><td>Multiclamp 700B Amplifier</td><td></td><td></td><td></td></tr><tr><td>Potassium chloride, BioXtra, &amp;ge;99.0%</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td></td></tr><tr><td>Potassium phosphate monobasic anhydrous &amp;ge;99%</td><td>Sigma-Aldrich</td><td>7778-77-0</td><td>Stored in Dessicator</td></tr><tr><td>Pump</td><td>Longer precision pump Co., Ltd</td><td>T-S113&amp;amp;JY10-14</td><td></td></tr><tr><td>Silicone oil</td><td>Sigma-Aldrich</td><td>63148-62-9</td><td></td></tr><tr><td>Sodium Bicarbonate, BioXtra, 99.5-100.5%</td><td>Sigma-Aldrich</td><td>144-55-8</td><td></td></tr><tr><td>Sodium Chloride, BioXtra, &amp;ge;99.5% (AT)</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium phosphate monobasic, powder</td><td>Sigma-Aldrich</td><td>7558-80-7</td><td></td></tr><tr><td>Sucrose, &amp;ge; 99.5% (GC)</td><td>Sigma-Aldrich</td><td>57-50-1</td><td></td></tr><tr><td>Temperature controller</td><td>Warner Instruments</td><td>TC-324C</td><td></td></tr><tr><td>Tungsten microelectrodes</td><td>FHC</td><td>20843</td><td></td></tr><tr><td>Urethane, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>51-79-6</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT-1200S</td><td></td></tr><tr><td>Water bath</td><td>Grant Instruments</td><td>SAP12</td><td></td></tr></tbody></table>

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 septotemporal axis, which is perpendicular to the transverse slices (Figure 1). Using recordings from the CA1 region of the hippocampus, we showed the presence of LTP that was not direction specific. There were no statistically significant differences in the recordings from the septal or temporal (Figure 2) side of the longitudinal hippocampal brain slice. We also showed the presence of LTP that was not layer specific; thus, recordings from both stratum radiatum and stratum oriens (Figure 2) showed successfully induced LTP in the longitudinal brain slice. We used D-AP5, an NMDAR antagonist to demonstrate that the LTP induced was dependent on NMDA receptors (Figure 3). What happens in vitro does not necessarily reflect in vivo conditions, so we investigated LTP in vivo. Figure 4a shows a schematic diagram of the stimulation and recording electrode positioned in the dorsal hippocampus along the longitudinal axis of CA1 region in vivo. The position of the electrodes used for the recording and stimulation was verified by lesion marks and crystal violet staining (Figure 4a). We demonstrated the presence of LTP in vivo in the longitudinal CA1 region (Figure 4b).
Using already established protocols for inducing LTD, we failed to successfully induce LTD both in vivo and in vitro (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59879/59879fig1.jpg" /> 
Figure 1. A schematic drawing of transverse and longitudinal hippocampal brain slices. This figure is adapted and modified from Sun et al. 201821. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59879/59879fig2.jpg" /> 
Figure 2. LTP in longitudinal slices. Synaptic responses at S.R. (a) or S.O. (b) in longitudinal slices are potentiated right after tetanus stimulation with both temporal and septal inputs (S.R./temporal (n = 12, c), S.R./septal (n = 12, c), S.O./temporal (n = 10, d), S.O./septal (n = 9, d). 
The n stands for the number of slices. Error bars represent SE. This figure is adapted and modified from Sun et al. 201821. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59879/59879fig3.jpg" /> 
Figure 3. NMDAR-dependent LTP in longitudinal slices. (a,b) LTP induction in temporal and septal direction is blocked by 50 &#956;M D-AP5 (temporal, n = 6, a) (septal, n = 5, b). (c,d) LTP induction in temporal and septal direction is also blocked by D-AP5. The n stands for slices. Error bars represent SE. This figure is adapted and modified from Sun et al. 201821. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59879/59879fig4.jpg" /> 
Figure 4. In vivo LTP in the interlamellar network. (a) A schematic drawing of recording and stimulation electrodes in anesthetized animals. The loci of recording (on the septal side of CA1) and stimulating electrodes (on the temporal side of CA1) were identified by lesion marks. (b) LTP is induced in the interlamellar connection by 100 Hz high frequency stimulation (HFS) (n = 10 mice). Color traces: before (black) and after (red) HFS. Error bars represent SE. This figure is adapted and modified from Sun et al. 201821. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59879/59879fig5.jpg" /> 
Figure 5. Absence of in vivo and in vitro LTD in Interlamellar CA1 network. (a) 1 Hz-pp LFS does not induce in vivo LTD. (b) 1 Hz pp-LTP, (c) 5 Hz LFS, and (d) 1 Hz LFS do not produce LTD on either the temporal or septal sides of longitudinal brain slice. while LTD is induced by 1 Hz pp-LFS in transverse slices: temporal (n = 8), septal (n = 11) and transverse (n = 6) with 1 Hz pp-LFS; temporal (n = 3) and septal (n = 3) with 5 Hz LFS; temporal (n = 3) and septal (n = 3) with 1 Hz LFS. The n stands for slices. Error bars represent SE. This figure is adapted and modified from Sun et al. 201821. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59879/59879fig6.jpg" /> 
Figure 6. Input-output curve presenting fEPSP slope in response to increasing stimulus input in hippocampal brain slice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59879/59879fig7.jpg" /> 
Figure 7. Surgical tools used for hippocampal isolation during in vitro brain slicing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59879/59879fig8.jpg" /> 
Figure 8. A longitudinal brain slice ready for recording. Stimulation electrode and recording pipette are inserted in the stratum radiatum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59879/59879fig9.jpg" /> 
Figure 9. A transverse hippocampal brain slice ready for recording. Stimulation electrode is inserted at Schaffer collateral CA3 region and recording pippette is inserted at CA1 region. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59879/59879fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Slicing Solution(mM)</td>
			<td>ACSF (mM)</td>
		</tr>
		<tr>
			<td>CaCl2.2H2O</td>
			<td>0.5</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>25</td>
			<td>25</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>7</td>
			<td>1</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>87</td>
			<td>125</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>1.3</td>
			<td>1.3</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>25</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>75</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Concentrations of compounds in brain slice and artificial cerebrospinal fluid solutions.

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 ...

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12.4K Views.  City University of Hong Kong. Langdurige synaptische plasticiteit, met name, langdurige potentiëring en langdurige depressie zijn op grote schaal onderzocht langs de transversale oriëntatie van de hippocampus. Er is ook naar gekeken langs de longitudinale oriëntatie. Echter, als het gaat om de longitudinale oriëntatie, is er zeer weinig aandacht besteed aan de longitudinale CA1-as van de hippocampus met betrekking tot synaptische plasticiteit.Een eerdere publicatie van ons lab hebben aangetoond dat de CA1 piram...

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

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

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

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

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

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

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Source: Whitebirch, A. C. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61704">Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity.</a> J. Vis. Exp. (2020)
The video demonstrates a procedure to record the local field potential (LFP) in mouse hippocampal-entorhinal cortex slices. Electrical stimulation is provided at the CA3 stratum radiatum of the hippocampus, which causes changes in the postsynaptic potential at the CA1 stratum pyramidale. The combined change in the membrane potential of CA1 neurons, termed the local field potential, is recorded.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Transcardial perfusion

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Recording Neuronal Field Excitatory Postsynaptic Potentials in Acute Hippocampal Slices

Source: Weng, W., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55936">Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure for setting up a brain slice in a submersion chamber to record synaptic activity. The process involves securing the slice on lens paper, maintaining a continuous flow of artificial cerebrospinal fluid (aCSF) to ensure neuronal viability, and accurately positioning the stimulation and recording electrodes relative to a specific brain region of interest. This setup allows for precise monitoring of baseline synaptic activity and the effects of electrical stimulation.

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

Recording CA3-CA1 Synaptic Responses in a Rat Hippocampal Slice

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

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

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

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