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.

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

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 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, ...

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 ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...