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 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.
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.
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
2. Rotor rod test
3. Inhibitory avoidance test
4. Dissection buffer
5. Artificial cerebrospinal fluid (aCSF)
6. Intracellular solutions
7. Slice preparation
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).
9. Current-clamp analysis
10. Voltage-clamp analysis
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 µM 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.
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<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<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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µm. F: Changes in the pipette current before touch (top) and at membrane rupture (bottom). Please click here to view a larger version of this figure.
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<0.05, **P<0.01 vs. untrained. Please click here to view a larger version of this figure.
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 µM). 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 ± SEM. **P<0.01 vs. untrained rats. Please click here to view a larger version of this figure.
Dissection buffer (Total 1L) | ||
NaH2PO4 • 2H2O | 0.195 g | 1.25 mmol/L |
KCl | 0.188 g | 2.5 mmol/L |
CaCl2 | 0.074 g | 0.5 mmol/L |
MgCl2 • 6H2O | 1.423 g | 7.0 mmol/L |
Choline chloride | 12.579 g | 90 mmol/L |
Ascorbic acid | 2.340 g | 11.6 mmol/L |
Pyruvic acid | 0.342 g | 3.1 mmol/L |
NaHCO3 | 2.100 g | 25 mmol/L |
Glucose | 4.500 g | 25 mmol/L |
Table 1: A recipe for dissection buffer
Artificial CSF (Total 1L) | ||
KCl | 0.186 g | 2.5 mmol/L |
NaCl | 6.700 g | 114.6 mmol/L |
NaH2PO4 •2H2O | 0.156 g | 1 mmol/L |
Glucose | 1.800 g | 10 mmol/L |
NaHCO3 | 2.184 g | 26 mmol/L |
1M MgCl2 | 4 mL | 4 mmol/L |
1M CaCl2 | 4 mL | 4 mmol/L |
Table 2: A recipe for artificial cerebrospinal fluid (CSF)
Intracellular solution for current clamp (Total 200 mL) | ||
KCl | 0.0746 g | 5 mmol/L |
K-Gluconate | 6.089 g | 130 mmol/L |
HEPES | 0.476 g | 10 mmol/L |
EGTA | 0.0456 g | 0.6 mmol/L |
1M MgCl2 | 500 µL | 2.5 mmol/L |
Na2 ATP | 0.4408 g | 4 mmol/L |
Na3 GTP | 0.0418 g | 0.4 mmol/L |
Na phosphocreatine | 0.510 g | 10 mmol/L |
Table 3: A recipe for an intracellular solution for current clamp recording
Intracellular solution for voltage clamp (Total 200 mL) | ||||
CsMeSO3 | 5.244 g | (5.814)* | 115 mmol/L | (127.5)* |
CsCl | 0.672 g | (0.252)* | 20 mmol/L | (7.5)* |
HEPES | 0.476 g | 10 mmol/L | ||
EGTA | 0.0456 g | 0.6 mmol/L | ||
1M MgCl2 | 500 µL | 2.5 mmol/L | ||
Na2 ATP | 0.4408 g | 4 mmol/L | ||
Na3 GTP | 0.0418 g | 0.4 mmol/L | ||
Na phosphocreatine | 0.510 g | 10 mmol/L | ||
* low Cl– concentration for miniature recordings |
Table 4: A recipe for an intracellular solution for voltage clamp recording
Parameters | untrained | IA trained | unpaired | walk through | |
mEPSC amplitude | Variance | 5.8 | 32.1 | 4.7 | 5.9 |
Standard deviation | 2.4 | 5.7 | 2.2 | 2.4 | |
Coefficient of variation | 0.189 | 0.326 | 0.177 | 0.190 | |
mIPSC amplitude | Variance | 17.1 | 56.7 | 31.8 | 20.7 |
Standard deviation | 4.1 | 7.5 | 5.6 | 4.5 | |
Coefficient of variation | 0.279 | 0.387 | 0.367 | 0.286 |
Table 5: The diversity of miniature excitatory and inhibitory postsynaptic current (mEPSC and mIPSC) amplitudes in inhibitory avoidance (IA)-trained rats
Parameters | untrained | IA trained | unpaired | walk through | |
mEPSC frequency | Variance | 278 | 2195 | 188 | 195 |
Standard deviation | 17 | 47 | 14 | 14 | |
Coefficient of variation | 0.902 | 1.198 | 0.893 | 0.874 | |
mIPSC frequency | Variance | 3282 | 27212 | 1385 | 5135 |
Standard deviation | 57 | 165 | 37 | 72 | |
Coefficient of variation | 1.195 | 1.006 | 0.955 | 0.836 |
Table 6: The diversity of miniature excitatory and inhibitory postsynaptic current (mEPSC and mIPSC) frequencies in inhibitory avoidance (IA)-trained rats
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 authors have nothing to disclose.
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.
Rota-Rod Treadmills | Med Associates Inc. | ENV577 | |
inhibitory avoidance box | Shinano Seisakusho | ||
Pentobarbital | Kyoritsu Seiyaku | ||
Blade | Nisshin EM Co., Ltd | LC05Z | |
Cardiac perfusion syringe | JMS Co., Ltd | JS-S00S | |
Vibratome | Leica Microsystems | VT-1200 | |
Horizontal puller | Sutter Instrument | Model P97 | |
Microfilm 34 gauge | World Precision Instruments, Inc | MF34G-5 | |
0.22 µm filter | Millipore | SLGVR04NL | |
Axopatch–1D amplifier | Axon Instruments | ||
Digidata 1440 AD board | Axon Instruments | ||
pCLAMP 10 software | Axon Instruments | ||
Upright Microscope | Olympus | BX51WI | |
CCD camera | Olympus | U-CMAD3 | |
Camera controller | Hamamatsu Photonics K.K. | C2741 | |
Stimulator | Nihon Kohden | SEN-3301 | |
Isolator | Nihon Kohden | SS-104J | |
Motorized manipulator | Sutter Instrument | MP-285 | |
Micromanipulator | Narishige | NMN-21 | |
Peristaltic Pump | Gilson, Inc | MINIPULS® 3 | |
Glass capillary | Narishige | GD-1.5 | |
Ag/AgCl electrode | World Precision Instruments, Inc | EP4 | |
Slice Anchor | Warner instruments | 64-0252 | |
Stimulus electrode | Unique Medical Co., Ltd | KU201-025B | |
Materials | Company | Catalog Number | Comments |
Dissection buffer/ artificial CSF |
|||
NaH2PO4 • 2H2O | Sigma-Aldrich Co. | C1426 | |
KCl | Wako Pure Chemical Industries | 163-03545 | |
CaCl2 | Wako Pure Chemical Industries | 039-00475 | |
MgCl2 • 6H2O | Wako Pure Chemical Industries | 135-00165 | |
Choline chloride | Sigma-Aldrich Co. | C7527 | |
Ascorbic acid | Wako Pure Chemical Industries | 190-01255 | |
Pyruvic acid Na | Wako Pure Chemical Industries | 199-03062 | |
NaHCO3 | Sigma-Aldrich Co. | 28-1850-5 | |
Glucose | Sigma-Aldrich Co. | 07-0680-5 | |
Materials | Company | Catalog Number | Comments |
Intracellular solution | |||
K-Gluconate | Sigma-Aldrich Co. | G4500 | |
HEPES | Wako Pure Chemical Industries | 346-01373 | |
EGTA | Wako Pure Chemical Industries | 348-01311 | |
Na2 ATP | Nacalai Tesque | 01072-24 | |
Na3 GTP | Sigma-Aldrich Co. | G-8877 | |
Na phosphocreatine | Sigma-Aldrich Co. | P-7936 | |
CsMeSO3 | Sigma-Aldrich Co. | C1426 | |
CsCl | Wako Pure Chemical Industries | 033-01953 | |
Materials | Company | Catalog Number | Comments |
Drugs in aCSF | |||
2-Chloroadenosine | Sigma-Aldrich Co. | C5134 | |
Picrotoxin | Sigma-Aldrich Co. | P-1675 | |
Tetrodotoxin | Wako Pure Chemical Industries | 207-15901 | |
CNQX | Sigma-Aldrich Co. | C239 | |
APV | Sigma-Aldrich Co. | A5282 |