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.

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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.

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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>
			<td style="width:100px;">5908-99-6</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 height="20" style="height:20px;">D-(+)-Glucose &#8805; 99.5% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>50-99-7</td>
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			<td height="20" style="height:20px;">Eyegel</td>
			<td>Dechra</td>
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			<td height="21" style="height:21px;width:163px;">Isoflurane</td>
			<td>RWD Life Sciences</td>
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			<td height="21" style="height:21px;">Magnesium chloride hexahydrate, BioXtra, &#8805;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>7791-18-6</td>
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			<td height="41" style="height:41px;width:163px;">Matrix electrodes, Tungsten</td>
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			<td height="41" style="height:41px;width:163px;">Multiclamp 700B Amplifier</td>
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			<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 height="40" style="height:40px;width:163px;">Sodium Bicarbonate, BioXtra, 99.5-100.5%</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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			<td height="40" style="height:40px;width:163px;">Tungsten microelectrodes</td>
			<td>FHC</td>
			<td align="right">20843</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 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 ...

investigating-long-term-synaptic-plasticity-interlamellar-hippocampus

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.3K Views.  City University of Hong Kong. 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.

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

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 directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

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

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

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

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

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

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

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 recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

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

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

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 dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

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 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.

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 experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

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