We would like to thank Dr. Paw-Min-Thein-Oo, Dr. Han-Thiri-Zin, and Mrs. H. Tsurutani for their technical assistance. This project was supported by Grants-in-Aid for Young Scientists (H.K. and Y.S.), Scientific Research B (D.M.), Scientific Research C (D.M.), and Scientific Research in Innovative Areas (D.M.), from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

All animal housing and surgical procedures were in accordance with the Guidelines for Animal Experimentation of Yamaguchi University School of Medicine and were approved by the Institutional Animal Care and Use Committee of Yamaguchi University.
1. Animals
<ol>
	<li>Use 4- to 5-week-old male Sprague-Dawley rats (postnatal 28 to 31 days of age).</li>
	<li>House the rats in individual plastic cages (40 cm &#215; 25 cm &#215; 25 cm) maintained at a constant temperature (23 &#176;C &#177; 1 &#176;C) under a 12-h light/dark cycle. Give rats ad libitum access to water and food.</li>
</ol>
2. Rotor rod test
<ol>
	<li>To investigate motor skill learning, subject each rat to the rotor rod test (rod diameter 7 cm; lane width 8.9 cm; fall height 26.7 cm) for 1 or 2 consecutive days (Figure 1A in Kida et al., 20167). Perform the task in a quiet, temperature-controlled room (23 &#177; 1 &#176;C). Do not disturb or handle rats prior to the test.</li>
	<li>Set the rotor rod to acceleration mode, which increases linearly from 4 rotations/min to 40 rotations/min (8 &#960;/min to 80 &#960;/min) in 5 min.</li>
	<li>Put the rat on the resting rotating rod. Confirm that all limbs are on the rod.</li>
	<li>Measure the latency to fall from the rotating rod to evaluate motor performance.</li>
	<li>Allow each rat 10 attempts (trials) with 30-s intervals.</li>
	<li>If the rat falls from the rotating rod, set it on the rod again after a 10-20 s interval.</li>
	<li>Sacrifice the rat with an overdose of pentobarbital (400 mg/kg) 30 min after the final trial. Inject untrained control rats with the same dose of anesthesia in their home cages.</li>
</ol>
3. Inhibitory avoidance test
<ol>
	<li>To investigate contextual learning, subject rats to an inhibitory avoidance (IA) test (Figure 1D in Mitsushima et al., 2011, 201310,11) Avoid any episodic experiences on the day of the experiment such as contact with others, cage changes or cleaning. Perform the task in a quiet, temperature-controlled room (23 &#177; 1 &#176;C). 
	NOTE: The IA training apparatus is a two-chambered acrylic box (length 33 cm; width 58 cm; height 33 cm). It has a lighted safe side and a dark shock side that are separated by a trap door (Figure 1D).</li>
	<li>Place the rat in the safe (lit) side of the illuminated box. Handle the rat gently without stress.</li>
	<li>Wait a short time (10 to 20 s) to acclimate the rat to the environment.</li>
	<li>Open the sliding door to allow the rat to enter the dark box at will.</li>
	<li>Measure the latency (s) before the rat enters the novel dark side of the box. The latency of the first trial represents the rat&#39;s performance before training.</li>
	<li>After entry into the dark side, close the door and apply a scrambled electrical foot shock (2 s, 1.6 mA) via electrical steel rods set into the floor of the box. Allow walk-through rats to explore the training apparatus for 1 min without being shocked. House unpaired rats in an illuminated shock cage for several days and suddenly give the shock without episodic experiences. Handle gently without stress in any groups.</li>
	<li>Keep each rat in the dark box for 10 s before returning it to the home cage.</li>
	<li>At 30 min after the foot shock, again place the rat into the lighted side of the box. Measure the latency to enter the dark side.</li>
	<li>Return the rat to the home cage.</li>
	<li>At 60 min after the shock, sacrifice the rat with an overdose of pentobarbital (400 mg/kg). Handle the rat gently and inject the anesthesia intraperitoneally. In untrained control rats, inject the anesthesia in their home cages without the experience described above.</li>
</ol>
4. Dissection buffer
<ol>
	<li>Dissolve crystals of 0.195 g NaH2PO4-2H2O, 0.188 g KCl, 0.074 g CaCl2, 1.423 g MgCl2-6H2O, and 12.579 g choline chloride in ultrapure water (900 mL to 950 mL). See Table 1.</li>
	<li>Dissolve crystals of 2.340 g ascorbic acid, 0.342 g pyruvic acid sodium salt, 2.100 g NaHCO3, and 4.500 g glucose.</li>
	<li>Add water up to 1000 mL. The range of osmolality will be between 290 mOsm/L and 300 mOsm/L. Adjust osmolality by adding ultrapure water, if it is over the range.</li>
	<li>Bubble the solution with 5% CO2/95% O2 gas mixture at ice-cold temperatures for 5 min before use.</li>
</ol>
5. Artificial cerebrospinal fluid (aCSF)
<ol>
	<li>Dissolve crystals of 0.186 g KCl, 6.700 g NaCl, and 0.156 g NaH2PO4-2H2O in ultrapure water (900 mL to 950 mL). See Table 2.</li>
	<li>Bubble with the gas mixture for 5 min.</li>
	<li>Dissolve crystals of 1.800 g glucose and 2.184 g NaHCO3 and then add 4 mL MgCl2 and 4 mL CaCl2 from 1 M stock solutions.</li>
	<li>Add water up to 1000 mL. The range of osmolality will be between 290 mOsm/L and 295 mOsm/L. Adjust osmolality by adding ultrapure water, if it is over the range.</li>
	<li>Bubble with the gas mixture prior to use.</li>
</ol>
6. Intracellular solutions
<ol>
	<li>For current-clamp recordings (Table 3), dissolve 0.0746 g KCl, 6.089 g K-gluconate, 0.476 g HEPES, 0.0456 g EGTA, and 500 &#181;L MgCl2 from 1 M stock solution in 180 mL ultrapure water (adjust pH to 7.2 with KOH).
	<ol>
		<li>Add 0.4408 g Na2-ATP, 0.0418 g Na3-GTP, and 0.510 g Na-phosphocreatine. Add water to 200 mL and adjust the pH to 7.35 with KOH.</li>
		<li>Adjust the osmolality to around 290 mOsm/L by adding ultrapure water.</li>
		<li>Store as 1-mL aliquots in the freezer (-30 &#176;C).</li>
	</ol>
	</li>
	<li>For voltage-clamp recordings (Table 4), dissolve 5.244 g CsMeSO3, 0.672 g CsCl, 0.476 g HEPES, 0.0456 g EGTA, and 500 &#181;L MgCl2 from 1 M stock solutions in 180 mL ultrapure water. Adjust the pH to 7.2 with CsOH. For mEPSP and mIPSP recordings, use modified concentration of 5.814 g CsMeSO3 and 0.252 g CsCl to adjust the reversal potential of the GABAA receptor response11.
	<ol>
		<li>Add 0.4408 g Na2-ATP, 0.0418 g Na3-GTP, and 0.510 g Na-phosphocreatine. Add water to 200 mL and adjust the pH to 7.35 with CsOH.</li>
		<li>Adjust the osmolality to around 290 mOsm/L by adding ultrapure water .</li>
		<li>Store as 1-mL aliquots in the freezer (-30 &#176;C).</li>
	</ol>
	</li>
</ol>
7. Slice preparation
<ol>
	<li>Prior to sacrifice, cool-down all dissection tools with crushed ice (Figure 2A). Add about 500 mL of cold water into the crushed ice container to increase the contact surface area. This procedure was described previously10,11,12. 
	NOTE: The tools here are: Large scissors, iris scissors, a spatula, a micro spatula, forceps, tweezers, a stainless steel 200-mL beaker, a blade for brain trimming, a 120-mL cardiac perfusion syringe filled with dissection buffer treated with the gas mixture, a silicone tube (20 cm) connected to a flattened 18-gauge needle, a stainless brain dissection stage (thickness = 3 mm, &#981; = 12 cm), and a mounting stage for the vibratome (&#981; = 5 cm).</li>
	<li>Sacrifice the rat 30 min after completing the behavioral paradigm by anesthetizing it with an overdose of pentobarbital (400 mg/kg bodyweight). Perform the slice preparation rapidly to ensure that the slices are as healthy as possible10,11,12. The brain extraction protocol meets all veterinary standards for our university.</li>
	<li>Fill a 120-mL syringe with ice-cold dissection buffer (Table 1) bubbled with a 5% CO2/95% O2 gas mixture. Remove any air bubbles prior to perfusion.</li>
	<li>After exposing the heart, insert the needle into the posterior part of the left ventricle.</li>
	<li>Perform transcardial perfusion of the brain manually using the syringe. Larger rats require more dissection buffer for perfusion. Submerge the brain with ice-cold dissection buffer for 5 min. Bubble the buffer continuously during the submersion.</li>
	<li>Trim the posterior side of the brain at an angle parallel to the dendritic orientation of the target cortical region using a blade. Since the brain is stand on dissection stage with the cut end bottom, the initial angle determines the angle of all subsequent brain slices. This step is critically important (Figure 2B). An incorrect angle may cut through the target pyramidal neurons. 
	NOTE: The tools here are: a blade for brain trimming, a filter paper (&#981; = 10 cm), a stainless brain dissection stage (thickness = 3 mm, &#981; = 12 cm), a spatula, a superglue, a dropper, and a mounting stage for the vibratome (&#981; = 5 cm).</li>
	<li>Cut 350-&#181;m thick coronal brain slices using a vibratome. Fill the dissection chamber with ice-cold buffer bubbled with a 5% CO2/95% O2 gas mixture (Figure 2C). Bubble the buffer continuously during the brain slice.</li>
	<li>Trim the periphery of the target area using iris scissors.</li>
	<li>Wash the trimmed slices gently in room temperature aCSF bubbled with 5% CO2/95% O2 (Table 2).</li>
	<li>Maintain the trimmed slices in an interface chamber until the recording is performed (Figure 2D and E). Incubation for 1 h in the chamber improves the condition of the cells, but the phenotypes change if the slices are incubated for more than 10 hours. Close the lid of the chamber to enclose the gases and the small liquid drops of aCSF.</li>
</ol>
8. Whole-cell patch clamp
NOTE: Whole-cell recordings require an amplifier and a low-pass filter that is set to a cutoff frequency of 5 kHz. The signals are digitized and stored in a PC. The stored data are analyzed offline (Figure 3A).
<ol>
	<li>Create glass electrodes using a horizontal puller. Fill the electrodes with a suitable solution (Tables 3 and 4) using a regular polyethylene 1-mL syringe attached to a fine glass tube and a 0.22-&#181;m filter.</li>
	<li>Prior to contact with the cell, maintain positive pressure and adjust the pipette current to zero.</li>
	<li>After forming a gigaohm seal, apply negative pressure to rupture the cell membrane (whole-cell configuration in Figure 3F).</li>
</ol>
9. Current-clamp analysis
<ol>
	<li>Properties of the cell membrane
	<ol>
		<li>Fill the patch recording pipettes with the intracellular solution for current-clamp recordings (Table 3). The resistance of the pipette is between 4 M&#937; and 7 M&#937; in the aCSF.</li>
		<li>After the membrane ruptures, hold the membrane voltage at -60 mV in V-CLAMP mode. Then, switch from &#34;bath&#34; mode to &#34;cell&#34; mode in the membrane test using software to measure the intrinsic cell properties such as membrane capacitance, resistance, and time constant.</li>
	</ol>
	</li>
	<li>Current injection study
	<ol>
		<li>After recording the intrinsic cell properties, switch the mode from V-CLAMP to TRACK (I = 0) /I-CLAMP NORMAL for the current injection. Note that the liquid junction potential should not be corrected10.</li>
		<li>Inject current into the cell for 300 ms. Change the intensity of the current stepwise from &#8722;100 pA to +550 pA with 50-pA increases. Count the number of spikes (action potentials) elicited by the current injections.</li>
		<li>Measure the minimum voltage needed to induce an action potential (this is the threshold voltage).</li>
		<li>Calculate the afterhyperpolarization amplitude as the difference between the voltage at spike initiation and the lowest voltage attained during afterhyperpolarization7.</li>
	</ol>
	</li>
</ol>
10. Voltage-clamp analysis
<ol>
	<li>The AMPA/NMDA ratio 
	NOTE: The AMPA/NMDA ratio is a conventional way to evaluate postsynaptic plasticity at glutamatergic excitatory synapses7,8,9,10,11. However, note that concomitant increases in both components may not change the ratio13.
	<ol>
		<li>Perfuse the recording chamber with physiological solution bubbled with the gas mixture and maintain the temperature at 22 &#176;C to 25 &#176;C. Add 0.1 mM picrotoxin to the solution to block the GABAA-mediated response and add 4 &#181;M 2-chloroadenosine to stabilize the evoked neural response14.</li>
		<li>Fill the patch recording pipettes with the intracellular solution for voltage-clamp recordings (Table 4). Check the resistance of the recording pipette in the aCSF. The resistance is between 4 M&#937; and 7 M&#937;.</li>
		<li>For recording in layer II/III pyramidal neurons in the M1, place a bipolar tungsten stimulating electrode 200 &#181;m to 300 &#181;m lateral to the cells to be recorded, below the pial surface in the region of the forelimb representation (2-mm lateral to the midline)15,16,17.</li>
		<li>For recording in a CA1 pyramidal neuron, place the stimulating electrode 200 &#181;m to 300 &#181;m lateral (Schaffer collateral fiber) or medial (temporoammonic pathway) to the cells that will be recorded (Figure 3B).</li>
		<li>Increase the stimulus intensity up to the synaptic response &#62; 10 pA.</li>
		<li>Calculate the AMPA/NMDA ratio as the ratio of the peak current measured at &#8722;60 mV to the current measured at +40 mV at 150 msec after the stimulus onset. Note that 50 to 100 traces should be averaged to calculate the ratio.</li>
	</ol>
	</li>
	<li>Miniature postsynaptic current recordings 
	Note: Miniature excitatory postsynaptic currents (mEPSCs) are thought to correspond to the responses elicited by the presynaptic release of a single vesicle of glutamate18. In contrast, miniature inhibitory postsynaptic currents (mIPSCs) are thought to correspond to the responses elicited by the presynaptic release of a single vesicle of GABA18. Increases in the amplitudes of mEPSCs and mIPSCs reflects postsynaptic transmission strengthening, while increases in the event frequency reflect increases in the number of functional synapses or the presynaptic release probability11.
	<ol>
		<li>Fill the patch recording pipette with modified intracellular solution (Table 4) to adjust the reversal potential of the GABAA receptor-mediated current to -60 mV.</li>
		<li>Add 0.5 &#181;M tetrodotoxin to the bath to block spontaneous action potentials.</li>
		<li>Hold the voltage at -60 mV to record the mEPSC events for 5 min.</li>
		<li>Change the holding potential to 0 mV to record mIPSC events for 5 min. Because M1 neurons show slightly higher reversal potential for AMPA receptor-mediated currents, the mIPSCs of M1 neurons are recorded at +15 mV with 0.1 mM APV.</li>
		<li>Wait for a few minutes for the current to stabilize.</li>
		<li>Record the mIPSC events for 5 min.</li>
		<li>Detect the miniature events using the software, and use events above 10 pA for the analysis. Count the number of mEPSCs or mIPSCs events for 5 min to determine the frequency. Average the amplitudes of the events to obtain the mean amplitude.</li>
		<li>Confirm whether bath treatment with 10 &#181;M CNQX or with 10 &#181;M bicuculline methiodide blocks the mEPSCs and mIPSCs events, respectively.</li>
	</ol>
	</li>
	<li>Paired-pulse analysis 
	NOTE: Presynaptic plasticity can be analyzed using paired-pulse analysis. An increase in the paired-pulse rate suggests a decrease in the presynaptic glutamate or GABA release probability7,10,11.
	<ol>
		<li>To analyze excitatory synapses, add 0.1 mM picrotoxin and record the response at -60 mV. Although we added 4 &#181;M 2-chloroadenosine to the bath, we need to keep in mind that the drug affects the presynaptic release probability14 .</li>
		<li>To analyze inhibitory synapses, add 0.1 mM APV and 4 &#181;M 2-chloroadenosine to the bath and record the response at 0 mV. In M1 neurons, record the response at +15 mV.</li>
		<li>Apply paired pulses with an inter-stimulus interval of 100 ms or 200 ms.</li>
		<li>Record 50-100 sequential traces at each holding potential and average the values.</li>
		<li>Calculate the paired pulse ratio as the ratio of the second peak to the first peak of the postsynaptic current.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+plasticity+within+highly+specific+neuronal+populations+identifies+a+unique+parcellation+of+motor+learning+in+the+adult+brain.">Structural plasticity within highly specific neuronal populations identifies a unique parcellation of motor learning in the adult brain.</a> Proc Natl Acad Sci U S A. 108 (6), 2545-2550 (2011).</li></ol>

The authors declare no conflicts of interest. We confirm that we have read the Journal's position on issues involved in ethical publication, and we affirm that this report is consistent with those guidelines. The funders had no role in the study design, data collection or analysis, the decision to publish, or the preparation of the manuscript.

The major limitation of the slice patch clamp technique is the recording in slice preparation, which may not reflect what happens in vivo. Although in vivo current-clamp analysis is more reliable, it is technically challenging to get sufficient data from conscious animals. Since each pyramidal neuron has different cellular properties, an adequate number of cells is needed to properly analyze differences in neurons after training. Moreover, voltage-clamp analysis requires continuous drug treatment with CNQX, APV, or bicuculline to determine the nature of the postsynaptic responses. To analyze the miniature responses induced by a single vesicle of glutamate or GABA, continuous treatment with tetrodotoxin is needed to block spontaneous action potentials. Although the recently developed multi-photon imaging technique is powerful for analyzing morphological changes at excitatory synapses19, a combined patch clamp technique is needed to analyze the function of synapses in vivo. It is currently quite difficult to analyze morphological changes at inhibitory synapses, since most inhibitory synapses do not form spines. At this time, the slice patch clamp would be the most suitable technique to analyze cell properties or the functions of excitatory/inhibitory synapses in trained animals.
Using current-clamp analysis (Figure 4), we recently reported motor learning-induced intrinsic plasticity in layer II/III neurons. Specifically, the 1-day trained rats showed a significant decrease in resting membrane potential and an increase in the spike threshold. The 2-day trained rats showed a significant increase in resting membrane potential that led to increased excitability. These results suggested that there were dynamic changes in the intrinsic plasticity of M1 layer II/III neurons in trained rats. Additional voltage-clamp analysis revealed an increase in the paired-pulse ratio in 1-day trained rats, suggesting that there was a transient decrease in the presynaptic GABA release probability7. It is therefore possible that disinhibition from GABA at the layer II/III synapses might trigger the resulting learning-induced plasticity in the M1. In support of this, slice preparation of the M1 requires bath treatment with a GABAA receptor blocker to induce LTP20.
Analysis of miniature postsynaptic potentials is a powerful way to detect synaptic plasticity in IA-trained animals. Sequential recording of mEPSCs and mIPSCs in a single CA1 neuron allows the analysis of the synaptic excitatory/inhibitory strength of each individual neuron. Since a single mE(I)PSC response is attributed to a single vesicle of glutamate or GABA, an increase in the mE(I)PSC amplitude suggests postsynaptic strengthening. Using mE(I)PSC analysis, we found individual differences in the strength of excitatory/inhibitory input into each CA1 neuron (Figure 5C). IA training clearly promoted diversity in synaptic strength, but this was not observed in other groups (Table 5).
Learning-induced synaptic diversity can be analyzed mathematically. By calculating the appearance probability of each point, data from each neuron can be converted to self-entropy (bit) using the information theory of Claude E. Shannon21. A point with high appearance probability (around the mean level) indicates low self-entropy, while a point with very rare probability (a deviated point) indicates high self-entropy. Compared with untrained rats, the self-entropy per neuron was clearly increased in IA-trained rats but not in unpaired or walk-through rats22. This analysis suggests that there was an increase in intra-CA1 information after the contextual learning.
The slice patch clamp technique can also be used for cued fear conditioning studies in the lateral amygdala9 and for sensory experience studies in the barrel cortex8. Moreover, this technique can be used with various other techniques for further investigations. For instance, the virus-mediated green fluorescent protein (GFP)-tagged gene delivery technique can be combined with the patch clamp technique to analyze the function of specific molecules. In addition, focal microinjection of a retrograde tracer can be used to visualize specific neurons that project to a specific area. Then, using the current-clamp technique, cell-specific properties can be analyzed in the visualized neurons23. Further, combining two-photon laser-scanning microscopy with two-photon laser uncaging of glutamate has been used to demonstrate spine-specific growth and the EPSC response in mouse cortical layer II/III pyramidal neurons19. Thus, the slice patch clamp technique is being improved by combining it with novel chemicals, gene delivery, and photo manipulation techniques.

The patch clamp technique, developed by Neher and Sakmann, has been widely used for electrophysiological experiments1. The whole-cell patch clamp technique2 can be used to record intracellular current or voltage using the gigaohm seal of the cell membrane. The current-clamp technique allows us to analyze differences in membrane properties such as resting potential, resistance, and capacitance3. The voltage-clamp technique allows us to analyze learning-induced synaptic plasticity at both excitatory and inhibitory synapses.
The primary motor cortex (M1) is a central region that is critical for making skilled voluntary movements. Previous electrophysiological studies demonstrated the development of long-term potentiation (LTP)-like plasticity in layer II/III excitatory synapses after skilled motor training4. Moreover, in vivo imaging studies further demonstrated the remodeling of M1 dendritic spines after a skilled reaching task5,6. However, learning-induced synaptic and intrinsic plasticity has not been shown in M1 neurons.
We recently reported that a rotor rod task promoted dynamic changes in glutamatergic and GABAergic synapses and altered the intrinsic plasticity in M1 layer II/III neurons7. Here we used the slice patch clamp technique to investigate learning-induced plasticity. This technique can also be used to investigate other types of experience-dependent plasticity in other brain regions. For example, sensory input into the barrel cortex can strengthen AMPA receptor-mediated excitatory input into layer II/III neurons8, and cued fear conditioning strengthens the excitatory inputs onto the lateral amygdala neurons, which is required for fear memory9. Moreover, contextual learning creates diversity in terms of excitatory and inhibitory synaptic input into hippocampal CA1 neurons10,11.

<table><tbody><tr><td>Rota-Rod Treadmills</td><td>Med Associates Inc.</td><td>ENV577</td><td></td></tr><tr><td>inhibitory avoidance box</td><td>Shinano Seisakusho</td><td></td><td></td></tr><tr><td>Pentobarbital</td><td>Kyoritsu Seiyaku</td><td></td><td></td></tr><tr><td>Blade</td><td>Nisshin EM Co., Ltd</td><td>LC05Z</td><td></td></tr><tr><td>Cardiac perfusion syringe</td><td>JMS Co., Ltd</td><td>JS-S00S</td><td></td></tr><tr><td>Vibratome</td><td>&amp;nbsp;Leica Microsystems</td><td>VT-1200</td><td></td></tr><tr><td>Horizontal puller</td><td>Sutter Instrument</td><td>Model P97</td><td></td></tr><tr><td>Microfilm 34 gauge</td><td>World Precision Instruments, Inc</td><td>MF34G-5</td><td></td></tr><tr><td>0.22 &amp;micro;m filter</td><td>Millipore</td><td>SLGVR04NL</td><td></td></tr><tr><td>Axopatch&amp;ndash;1D amplifier&amp;nbsp;</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Digidata 1440 AD board</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>pCLAMP 10 software</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Upright Microscope</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>CCD camera</td><td>Olympus</td><td>U-CMAD3</td><td></td></tr><tr><td>Camera controller</td><td>Hamamatsu Photonics K.K.</td><td>C2741</td><td></td></tr><tr><td>Stimulator</td><td>Nihon Kohden</td><td>SEN-3301</td><td></td></tr><tr><td>Isolator</td><td>Nihon Kohden</td><td>SS-104J</td><td></td></tr><tr><td>Motorized manipulator</td><td>Sutter Instrument</td><td>MP-285</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>NMN-21</td><td></td></tr><tr><td>Peristaltic Pump&amp;nbsp;</td><td>Gilson, Inc</td><td>MINIPULS&amp;reg; 3</td><td></td></tr><tr><td>Glass capillary</td><td>Narishige</td><td>GD-1.5</td><td></td></tr><tr><td>Ag/AgCl electrode&amp;nbsp;</td><td>World Precision Instruments, Inc</td><td>EP4</td><td></td></tr><tr><td>Slice Anchor</td><td>Warner instruments</td><td>64-0252</td><td></td></tr><tr><td>Stimulus electrode</td><td>Unique Medical Co., Ltd</td><td>KU201-025B</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Dissection buffer/&lt;br/&gt; artificial CSF&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; &amp;bull; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich Co.</td><td>C1426</td><td></td></tr><tr><td>KCl</td><td>Wako Pure Chemical Industries</td><td>163-03545</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Wako Pure Chemical Industries</td><td>039-00475</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;bull; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako Pure Chemical Industries</td><td>135-00165</td><td></td></tr><tr><td>Choline chloride&amp;nbsp;</td><td>Sigma-Aldrich Co.</td><td>C7527</td><td></td></tr><tr><td>Ascorbic acid</td><td>Wako Pure Chemical Industries</td><td>190-01255</td><td></td></tr><tr><td>Pyruvic acid Na</td><td>Wako Pure Chemical Industries</td><td>199-03062</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich Co.</td><td>28-1850-5</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich Co.</td><td>07-0680-5</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Intracellular solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>K-Gluconate</td><td>Sigma-Aldrich Co.</td><td>G4500</td><td></td></tr><tr><td>HEPES</td><td>Wako Pure Chemical Industries</td><td>346-01373</td><td></td></tr><tr><td>EGTA</td><td>Wako Pure Chemical Industries</td><td>348-01311</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt; ATP</td><td>Nacalai Tesque</td><td>01072-24</td><td></td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt; GTP</td><td>Sigma-Aldrich Co.</td><td>G-8877</td><td></td></tr><tr><td>Na phosphocreatine</td><td>Sigma-Aldrich Co.</td><td>P-7936</td><td></td></tr><tr><td>CsMeSO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich Co.</td><td>C1426</td><td></td></tr><tr><td>CsCl</td><td>Wako Pure Chemical Industries</td><td>033-01953</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Drugs in aCSF&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Chloroadenosine</td><td>Sigma-Aldrich Co.</td><td>C5134</td><td></td></tr><tr><td>Picrotoxin&amp;nbsp;</td><td>Sigma-Aldrich Co.</td><td>P-1675</td><td></td></tr><tr><td>Tetrodotoxin</td><td>Wako Pure Chemical Industries</td><td>207-15901</td><td></td></tr><tr><td>CNQX&amp;nbsp;</td><td>Sigma-Aldrich Co.</td><td>C239</td><td></td></tr><tr><td>APV</td><td>Sigma-Aldrich Co.</td><td>A5282</td><td></td></tr></tbody></table>

slice patch clamp technique for analyzing learning-induced plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

As we described recently7, rotor rod training (Figure 1A) induced dynamic changes in the intrinsic plasticity of the M1 layer II/III pyramidal neurons. Measuring the latency until the rats fall from the rotating rod allows us to estimate the skilled learning performance of the rat. Longer latency indicates better motor performance. On the day 1 of training, the rats improved their rotor rod performance until the trial ended. On day 2, the rats attained nearly asymptotic levels in the averaged session scores (Figure 1B). Compared with the latency at the first trial, post-hoc analysis showed significant improvements at the final trials on the training days (Figure 1C).
Figure 4A shows an example of current-clamp analysis in which the neuronal properties changed after motor skill learning. Injections of 400 pA and 500 pA currents were needed to induce action potentials in the untrained group and in the 1-day trained rats, respectively. In contrast, injection of only a 150 pA current was sufficient to elicit action potentials in the 2-day trained rats. The relationship between the current intensity and the number of action potentials is shown in Figure 4B. As little as 50 pA current was sufficient to elicit spikes in 2-day trained rats; in contrast, 1-day trained rats responded with fewer action potentials than untrained rats to 350 pA and higher currents. Moreover, Figure 4C shows that 1-day trained rats showed lower resting potential, higher spike threshold, and deeper afterhyperpolarization, whereas 2-day trained rats showed higher resting potential (Figure 4C) and membrane resistance (Figure 4D).
As we described previously11, IA training (Figure 1D) induced postsynaptic plasticity at excitatory and inhibitory synapses of the hippocampal CA1 neurons. By measuring the latency in the light box, we could estimate the contextual learning performance of the rat. Figure 1E shows the results of the IA task. After the paired electric shock, the rats learn to avoid the dark side of the box and stay in the lighted side, which usually they would not prefer. The tendency to avoid the dark side therefore indicates the acquisition of contextual memories.
Figure 5 shows an example of voltage-clamp analysis in which miniature postsynaptic currents were dramatically changed after contextual learning. To investigate learning-induced plasticity, spontaneous AMPA-mediated mEPSCs and GABAA-mediated mIPSCs were sequentially recorded in the presence of 0.5 &#181;M tetrodotoxin (Figure 5A and B). As shown on two-dimensional plots (Figure 5C), each CA1 neuron had different mean amplitudes for mEPSCs and mIPSCs. Although the amplitudes were low and showed a narrow distribution range in untrained, unpaired, and walk-through rats, those were diverse in IA-trained rats (Table 5). ANOVA followed by post-hoc analysis showed a significant increase in the mean amplitudes of mEPSC and mIPSC in IA-trained rats (Figure 5E), suggesting learning-induced postsynaptic plasticity in the CA1 neurons.
Moreover, each CA1 neuron exhibited different mEPSC and mIPSC frequencies (Figure 5D). Although the frequencies were low and showed a narrow distribution range in untrained, unpaired, and walk-through rats, those were diverse in IA-trained rats (Table 6). ANOVA followed by post-hoc analysis showed a significant increase in the frequencies of the mEPSC and mIPSC events in IA-trained rats (Figure 5F). There are two possible interpretations of these results. The first is that contextual learning increased the number of functional synapses of the neurons. The other is that contextual learning increased the presynaptic release probability of glutamate and GABA.
To further examine presynaptic plasticity, we also conducted paired-pulse stimulations, as reported previously10,11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55876/55876fig1v2.jpg" /> 
Figure 1: Learning performance after training. 
A: The experimental design shows the rotor rod training and coronal brain slice. B: The mean latency to fall from the accelerating rotor rod barrel. C: The mean latency to fall off of the rod on the first and the final trials on training days 1 and 27. **P&#60;0.01 vs. first trial. D: Schema of the inhibitory avoidance (IA) task and coronal brain slice. E: The mean latency to enter the dark box before and after IA training11. **P&#60;0.01 vs. before IA training. The numbers by the coronal sections indicate the distance anterior to the bregma in mm. The number of animals is shown at the bottom of the bars. Error bars indicate SEM. <a href="https://cloudflare.jove.com/files/ftp_upload/55876/55876fig1v2large.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/55876/55876fig2.jpg" /> 
Figure 2: Slice procedures. 
A: Photographs show the preparation of acute brain slices. The dissection tools were cooled in crushed ice prior to use. B: Brain dissection and trimming. Note that the angle of trimming on the posterior side must be oriented in parallel with the dendritic orientation. C: Slicing the brain in a vibratome chamber. The brain is bathed in dissection buffer and bubbled continuously with a 5% CO2/95% O2 gas mixture. D: An interface chamber made of two plastic food containers and a silicone tube. The chamber was filled with artificial CSF and bubbled continuously with the gas mixture. E: Brain slices were placed on wet filter paper in the chamber. Bar = 5 mm. <a href="https://cloudflare.jove.com/files/ftp_upload/55876/55876fig2large.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/55876/55876fig3.jpg" /> 
Figure 3: Patch clamp procedures. 
A: The patch-clamp system used to record electrical signals from a neuron. The location of the stimulating and recording electrodes in the layer II/III neurons are shown in the rat motor cortex. B: To analyze the Schaffer synapses of a CA1 pyramidal neuron, a stimulating electrode was placed at the stratum radiatum. To analyze temporoammonic synapses, a stimulating electrode was placed at the stratum moleculare. Representative traces of evoked AMPA and NMDA receptor-mediated excitatory postsynaptic currents in the same CA1 neuron are shown. C: A slice anchor was used to stabilize the slice in the recording chamber. D: A representation map in the motor cortex, based on the published papers15,16,17. ML = midline. E: IR-DIC micrographs of M1 layer II/III neurons before (upper) and during the recording (lower). Bar = 10 &#181;m. F: Changes in the pipette current before touch (top) and at membrane rupture (bottom). <a href="https://cloudflare.jove.com/files/ftp_upload/55876/55876fig3large.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/55876/55876fig4.jpg" /> 
Figure 4: Representative results of current-clamp analysis7. 
A: Representative traces of action potentials recorded after induction with current injections. B: Relationships between the mean current input (pA) vs. action potential output (number of spikes) in brain slices from untrained (open bars), 1-day trained (gray bars), and 2-day trained rats (filled bars). C: Resting potential, threshold, and afterhyperpolarization of the layer II/III neurons. D: Membrane resistance and series resistance of the neurons. We used 9 - 10 rats in each group. The number of cells is shown within each bar. Error bars indicate the SEM. *P&#60;0.05, **P&#60;0.01 vs. untrained. <a href="https://cloudflare.jove.com/files/ftp_upload/55876/55876fig4large.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/55876/55876fig5.jpg" /> 
Figure 5: Representative results of the voltage-clamp analysis11. 
Representative traces of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) in untrained (A) and inhibitory avoidance (IA)-trained rats (B). mEPSCs at -60 mV and mIPSCs at 0 mV were measured sequentially in the same CA1 pyramidal neuron in the presence of tetrodotoxin (0.5 &#181;M). Vertical bar = 20 pA, horizontal bar = 200 msec. C: Two-dimensional plots of the mean mE(I)PSC amplitudes in untrained, IA-trained, unpaired, and walk-through rats. D: Two-dimensional plots of the mE(I)PSC frequencies in the 4 groups. Note that each CA1 neuron exhibited different mean mE(I)PSC amplitudes and frequencies. IA training not only strengthened the mean amplitudes (E) but also increased the frequencies of the mE(I)PSC events (F). We used 4 - 6 rats in each group. The number of cells is shown at the bottom of the bars. Red plus signs (C, D) and bars with vertical lines (E, F) indicate the mean &#177; SEM. **P&#60;0.01 vs. untrained rats. <a href="https://cloudflare.jove.com/files/ftp_upload/55876/55876fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Dissection buffer (Total 1L)</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 &#8226; 2H2O</td>
			<td>0.195 g</td>
			<td>1.25 mmol/L</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.188 g</td>
			<td>2.5 mmol/L</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.074 g</td>
			<td>0.5 mmol/L</td>
		</tr>
		<tr>
			<td>MgCl2 &#8226; 6H2O</td>
			<td>1.423 g</td>
			<td>7.0 mmol/L</td>
		</tr>
		<tr>
			<td>Choline chloride&#160;</td>
			<td>12.579 g</td>
			<td>90 mmol/L</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>2.340 g</td>
			<td>11.6 mmol/L</td>
		</tr>
		<tr>
			<td>Pyruvic acid</td>
			<td>0.342 g</td>
			<td>3.1 mmol/L</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>2.100 g</td>
			<td>25 mmol/L</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>4.500 g</td>
			<td>25 mmol/L</td>
		</tr>
	</tbody>
</table>
Table 1: A recipe for dissection buffer
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Artificial CSF (Total 1L)</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.186 g</td>
			<td>2.5 mmol/L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>6.700 g</td>
			<td>114.6 mmol/L</td>
		</tr>
		<tr>
			<td>NaH2PO4 &#8226;2H2O</td>
			<td>0.156 g</td>
			<td>1 mmol/L</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1.800 g</td>
			<td>10 mmol/L</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>2.184 g</td>
			<td>26 mmol/L</td>
		</tr>
		<tr>
			<td>1M MgCl2</td>
			<td>4 mL</td>
			<td>4 mmol/L</td>
		</tr>
		<tr>
			<td>1M CaCl2</td>
			<td>4 mL</td>
			<td>4 mmol/L</td>
		</tr>
	</tbody>
</table>
Table 2: A recipe for artificial cerebrospinal fluid (CSF)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Intracellular solution for current clamp (Total 200 mL)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.0746 g</td>
			<td>5 mmol/L</td>
		</tr>
		<tr>
			<td>K-Gluconate</td>
			<td>6.089 g</td>
			<td>130 mmol/L</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>0.476 g</td>
			<td>10 mmol/L</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.0456 g</td>
			<td>0.6 mmol/L</td>
		</tr>
		<tr>
			<td>1M MgCl2</td>
			<td>500 &#181;L</td>
			<td>2.5 mmol/L</td>
		</tr>
		<tr>
			<td>Na2 ATP</td>
			<td>0.4408 g</td>
			<td>4 mmol/L</td>
		</tr>
		<tr>
			<td>Na3 GTP</td>
			<td>0.0418 g</td>
			<td>0.4 mmol/L</td>
		</tr>
		<tr>
			<td>Na phosphocreatine</td>
			<td>0.510 g</td>
			<td>10 mmol/L</td>
		</tr>
	</tbody>
</table>
Table 3: A recipe for an intracellular solution for current clamp recording
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Intracellular solution for voltage clamp (Total 200 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>CsMeSO3</td>
			<td>5.244 g</td>
			<td>(5.814)*</td>
			<td>115 mmol/L</td>
			<td>(127.5)*</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>0.672 g</td>
			<td>(0.252)*</td>
			<td>20 mmol/L</td>
			<td>(7.5)*</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>0.476 g</td>
			<td></td>
			<td>10 mmol/L</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.0456 g</td>
			<td></td>
			<td>0.6 mmol/L</td>
			<td></td>
		</tr>
		<tr>
			<td>1M MgCl2</td>
			<td>500 &#181;L</td>
			<td></td>
			<td>2.5 mmol/L</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2 ATP</td>
			<td>0.4408 g</td>
			<td></td>
			<td>4 mmol/L</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3 GTP</td>
			<td>0.0418 g</td>
			<td></td>
			<td>0.4 mmol/L</td>
			<td></td>
		</tr>
		<tr>
			<td>Na phosphocreatine</td>
			<td>0.510 g</td>
			<td></td>
			<td>10 mmol/L</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="5">* low Cl- concentration for miniature recordings</td>
		</tr>
	</tbody>
</table>
Table 4: A recipe for an intracellular solution for voltage clamp recording
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameters</td>
			<td></td>
			<td>untrained</td>
			<td>IA trained</td>
			<td>unpaired</td>
			<td>walk through</td>
		</tr>
		<tr>
			<td>mEPSC amplitude</td>
			<td>Variance</td>
			<td>5.8</td>
			<td>32.1</td>
			<td>4.7</td>
			<td>5.9</td>
		</tr>
		<tr>
			<td></td>
			<td>Standard deviation</td>
			<td>2.4</td>
			<td>5.7</td>
			<td>2.2</td>
			<td>2.4</td>
		</tr>
		<tr>
			<td></td>
			<td>Coefficient of variation</td>
			<td>0.189</td>
			<td>0.326</td>
			<td>0.177</td>
			<td>0.190</td>
		</tr>
		<tr>
			<td>mIPSC amplitude</td>
			<td>Variance</td>
			<td>17.1</td>
			<td>56.7</td>
			<td>31.8</td>
			<td>20.7</td>
		</tr>
		<tr>
			<td></td>
			<td>Standard deviation</td>
			<td>4.1</td>
			<td>7.5</td>
			<td>5.6</td>
			<td>4.5</td>
		</tr>
		<tr>
			<td></td>
			<td>Coefficient of variation</td>
			<td>0.279</td>
			<td>0.387</td>
			<td>0.367</td>
			<td>0.286</td>
		</tr>
	</tbody>
</table>
Table 5: The diversity of miniature excitatory and inhibitory postsynaptic current (mEPSC and mIPSC) amplitudes in inhibitory avoidance (IA)-trained rats
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameters</td>
			<td></td>
			<td>untrained</td>
			<td>IA trained</td>
			<td>unpaired</td>
			<td>walk through</td>
		</tr>
		<tr>
			<td>mEPSC frequency</td>
			<td>Variance</td>
			<td>278</td>
			<td>2195</td>
			<td>188</td>
			<td>195</td>
		</tr>
		<tr>
			<td></td>
			<td>Standard deviation</td>
			<td>17</td>
			<td>47</td>
			<td>14</td>
			<td>14</td>
		</tr>
		<tr>
			<td></td>
			<td>Coefficient of variation</td>
			<td>0.902</td>
			<td>1.198</td>
			<td>0.893</td>
			<td>0.874</td>
		</tr>
		<tr>
			<td>mIPSC frequency</td>
			<td>Variance</td>
			<td>3282</td>
			<td>27212</td>
			<td>1385</td>
			<td>5135</td>
		</tr>
		<tr>
			<td></td>
			<td>Standard deviation</td>
			<td>57</td>
			<td>165</td>
			<td>37</td>
			<td>72</td>
		</tr>
		<tr>
			<td></td>
			<td>Coefficient of variation</td>
			<td>1.195</td>
			<td>1.006</td>
			<td>0.955</td>
			<td>0.836</td>
		</tr>
	</tbody>
</table>
Table 6: The diversity of miniature excitatory and inhibitory postsynaptic current (mEPSC and mIPSC) frequencies in inhibitory avoidance (IA)-trained rats

Watch this Scientific Journal Video about Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity at JoVE.com

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

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Research

JoVE Journal

Neuroscience

15.2K Views.  Yamaguchi University Graduate School of Medicine. The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

Video: Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Biology

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

Patch Clamp Electrophysiology

Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One extremely useful technique for investigating the biophysical properties of these channels is called patch clamp recording. In this method, neuroscientists place a polished glass micropipette against a cell and apply suction to form a high resistance seal. This process isolates a small &#8220;patch&#8221; of membrane that contains one or more ion channels. Using an electrode within the micropipette, researchers can &#8220;clamp&#8221; or control the electrical properties of the membrane, which is important for analysis of channel activity. The electrode also allows for changes in the voltage across the membrane, or the flow of ions through the membrane, to be recorded. This video begins with an overview of the principles behind patch clamp electrophysiology, an introduction to the necessary equipment, and descriptions of the various patch configurations, including whole cell, cell-attached, perforated, inside-out, and outside-out patches. Next, the key steps of a typical whole-cell patch clamp experiment are outlined, in which a current-voltage (IV) curve is generated. Finally, applications of patch clamp recording are provided to demonstrate how the biophysical properties of ion channels, cell excitability, and neuroactive compounds are evaluated in neurophysiology labs today.

Patch Clamp Electrophysiology: Principle &amp; Applications

Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One ...

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Advanced Biology

Patch Clamp

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively and negatively charged ions across the cell membrane. One competent method to record current flowing across the whole cell or single ion channel is the patch-clamp technique.
In this method, a glass micropipette containing electrolyte solution is tightly sealed against a small portion of the cell membrane. As a result, a patch of the cell membrane is electrically isolated, ensuring that the ions moving across the channels flow only into the micropipette. Further, this tight sealing of the cell membrane prevents ions from escaping into the bath solution in which the cell is suspended.
Different Patch-clamp Methods:
Depending upon the research interest, several variations of the patch-clamp method can be used for measuring the cell&#39;s biophysical properties. For example, researchers can clamp or control the membrane voltage and measure the current passing through it. Alternatively, they can clamp the current and measure any variation in the membrane voltage.
In cell-attached mode, the membrane patch containing a single or a few ion channels remains intact. As a result, the current flowing through the membrane patch alone can be measured. In contrast, the whole-cell method involves disruption of the membrane patch by briefly applying strong suction. Consequently, the interior of the pipette becomes continuous with the cytoplasm. This mode enables the measurement of electrical current and voltage from the entire cell.
Another patch-clamp method requires gentle retraction of the attached pipette. As a result, the membrane patch is excised without affecting the tight seal. In this inside-out configuration, the intracellular portion of the membrane is exposed to the bath solution, allowing the study of intracellular factors affecting the channel functions.

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively ...

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Cell Biology

Unit 1

Channels and the Electrical Properties of Membranes

KEY TERMS AND CONCEPTS

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice

In this procedure, transfer a brain slice to the recording chamber. Anchor the slice at the bottom of the recording chamber with a platinum ring, and ensure that the slice is of good quality with a smooth, even surface. Then, select a neuron with a dendrite extending over a long distance in the same plane, and ensure that the dendrite of interest can be followed to a well-defined soma. Next, fill the patch pipette with electrode solution.
To patch the dendrite in cell-attached mode, identify and focus on a portion of the dendrite. Apply positive pressure to the pipette, and lower it using the micromanipulator. Then, position the pipette close to the membrane, and adjust the pressure in order to create a small dimple. Subsequently, release the pressure on the pipette tip and patch the dendrite while controlling the pipette resistance. Try to obtain a seal resistance larger than 1 giga ohm, at best between 3 and 10 giga ohms for cell-attached recordings.
Afterward, retract the pipette away from the membrane by a couple of micrometers to avoid deformation of the dendrite. The action currents seen here represent the spontaneous action potentials of nigral neurons. To suppress the action currents, apply calcium and sodium channel blockers to ACSF. Then, apply a voltage step of negative 90 millivolts from a holding potential of 0 millivolts to evoke the hyperpolarization-activated cation current.

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Electrophysiology Techniques

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Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices

In this step, place one brain slice in the center of the recording chamber. Stabilize the brain slice with a slice holdown or a harp. To detect the fluorescent cell, find the area of interest under the four times magnification. Then, move the microscope stage by turning on &#34;click-to-move&#34; mode, and click the center of the area of interest. Next, switch to the high magnification lens and adjust the focus by moving the microscope in the z-axis using RF on the keypad.
The software directs the microscope in the camera to take a series of images at different depths. Then, these images are subjected to computer vision processing to find fluorescently labeled cells. Click the Detect Cell button on the main column, unit zero. A list of cell coordinates will appear in the memory position&#39;s GUI. Remove undesired cells from the list by clicking the X button next to the coordinates.
Then, click on the cell of interest. A yellow dot with a number will appear on the cell, and the coordinates of the cell will appear in the memory position&#39;s GUI. To perform secondary calibration in order to detect pipette offset coordinates, fill one-third of a glass pipette with internal solution. Then, load the pipette onto the pipette holder attached to the head stage.
At low magnification, use one and two on the keypad to switch between unit one and unit two. Then, bring the pipette into the visual field and adjust the focus using the keypad.
Next, load the primary calibration by clicking on Load Calibration. Switch the microscope lens to high magnification, and click 40 times on the main GUI. Bring the pipette tip to the center. Then, click the secondary calibration button on the main GUI under the unit that is in use. To patch a target cell, click on the Patch Control button to open the patch control GUI. Click the Go To button next to the cell of interest on the coordinate list in the memory position GUI.
Subsequently, click on the CTM button of the unit in the main GUI to enable movement. Click on the cell of interest to move the pipette tip to the cell.
Next, use the Unit 1 Selected button to switch the input signal between the two units. Click on the Patch button on the patch control GUI. Automatic patching will begin, and the pressure and resistance can be monitored on the patch control GUI.
The automatic patching algorithm monitors resistance and controls pressure through a series of logic loops to form a gigaseal. A pop-up window notifies the formation of a gigaseal.
The system utilizes resistance change to recognize cell surface contact. In a situation that the cell surface contact is not detected in time, use the Next button to advance patching stage while remaining in the same automatic patching trial.
Manipulate the automatic process at any point by clicking on the respective buttons on the patch control GUI. Then, select Yes to break in with combined zap and suction. Then, save the experiment patch log.