motor training.pdf

Cerebral Cortex, August 2016;26: 3494–3507 doi:10.1093/cercor/bhw134 Advance Access Publication Date: 18 May 2016 Original Article ORIGINAL ARTICLE Motor Training Promotes Both Synaptic and Intrinsic Plasticity of Layer II/III Pyramidal Neurons in the Primary Motor Cortex Hiroyuki Kida1, Yasumasa Ts...

Cerebral Cortex, August 2016;26: 3494–3507 doi:10.1093/cercor/bhw134 Advance Access Publication Date: 18 May 2016 Original Article ORIGINAL ARTICLE Motor Training Promotes Both Synaptic and Intrinsic Plasticity of Layer II/III Pyramidal Neurons in the Primary Motor Cortex Hiroyuki Kida1, Yasumasa Tsuda1, Nana Ito1, Yui Yamamoto2, Yuji Owada2, Yoshinori Kamiya3, and Dai Mitsushima1 1 Department of Physiology, 2Department of Organ Anatomy, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan and 3Uonuma Institute of Community Medicine, Niigata University Medical and Dental Hospital, 4132 Urasa, Minami-uonuma, Niigata 949-7302, Japan Address correspondence to Dai Mitsushima, Department of Physiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, 755-8505, Japan. Email: [email protected] Abstract Motor skill training induces structural plasticity at dendritic spines in the primary motor cortex (M1). To further analyze both synaptic and intrinsic plasticity in the layer II/III area of M1, we subjected rats to a rotor rod test and then prepared acute brain slices. Motor skill consistently improved within 2 days of training. Voltage clamp analysis showed significantly higher α-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl--aspartate (AMPA/NMDA) ratios and miniature EPSC amplitudes in 1-day trained rats compared with untrained rats, suggesting increased postsynaptic AMPA receptors in the early phase of motor learning. Compared with untrained controls, 2-days trained rats showed significantly higher miniature EPSC amplitude and frequency. Paired-pulse analysis further demonstrated lower rates in 2-days trained rats, suggesting increased presynaptic glutamate release during the late phase of learning. One-day trained rats showed decreased miniature IPSC frequency and increased paired-pulse analysis of evoked IPSC, suggesting a transient decrease in presynaptic γ-aminobutyric acid (GABA) release. Moreover, current clamp analysis revealed lower resting membrane potential, higher spike threshold, and deeper afterhyperpolarization in 1-day trained rats—while 2-days trained rats showed higher membrane potential, suggesting dynamic changes in intrinsic properties. Our present results indicate dynamic changes in glutamatergic, GABAergic, and intrinsic plasticity in M1 layer II/III neurons after the motor training. Key words: AMPA receptor, GABA, glutamic acid, motor learning Introduction The primary motor cortex (M1) is considered a central region required for skilled voluntary movements. During voluntary movements, neurons of the M1 vigorously discharge to encode various parameters, such as force (Evarts 1968), direction (Georgopoulos et al. 1982), and speed of spontaneous movements (Moran and Schwartz 1999). M1 neurons form a glutamatergic/γ-aminobutyric acidergic (GABAergic) neural circuit (Kaneko 2013), and synaptic transmission efficacy can be altered through motor experience. For example, forelimb motor training reportedly strengthens horizontal connections in M1 layer II/III (Rioult-Pedotti et al. 1998, 2000). In vivo imaging studies further demonstrated structural remodeling of dendritic spines of the M1 pyramidal neurons after skilled motor tasks, suggesting the learning-dependent © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Motor Training-Dependent Neocortical Plasticity plasticity of the M1 (Xu et al. 2009; Yang et al. 2009; Yu and Zuo 2011; Ma et al. 2016). Dendritic spines express glutamate receptors on their postsynaptic surface (Shi et al. 1999; Takumi et al. 1999). Activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)type glutamate receptors induces fast excitatory transmission enabling memory and enhancement of task-related behavior, and N-methyl--aspartate (NMDA) receptor activation is implicated in maintenance of spatial memory and associative learning (Riedel et al. 2003). Studies using virus-mediated in vivo gene delivery with in vitro patch-clamp recordings have also demonstrated that AMPA receptor delivery into the synapses is involved in synaptic strengthening (Malinow and Malenka 2002; Takahashi et al. 2003). Recently, we further showed that AMPA receptor delivery into CA1 synapses is required for episodic memory (Mitsushima et al. 2011). Since the LTP-like plasticity of M1 contributes to motor skill retention (Rioult-Pedotti et al. 2000; Cantarero et al. 2013), we hypothesized that an increase of postsynaptic AMPA receptors leads to synaptic strengthening among layer II/III neurons. Most research regarding plasticity focuses on excitatory synapses, but GABAergic inhibitory synapses are also strengthened by frequent presynaptic fiber stimulation (Caillard et al. 1999; Kurotani et al. 2008). Experience-dependent plasticity of GABAergic synapses was first reported in the hippocampus (Cui et al. 2008). We further demonstrated that contextual memory requires both AMPA and GABAA receptor-mediated postsynaptic plasticity, forming a wide diversity of excitatory/inhibitory inputs at hippocampal CA1 synapses (Mitsushima et al. 2013). However, motor learning rapidly reduces local GABA concentration in the human sensorimotor cortex (Floyer-Lea et al. 2006), suggesting that skilled motor learning may reduce GABAergic inhibitory transmission in the M1. In the present study, we further examined motor training-dependent plasticity at GABAergic synapses, as well as the balance of excitatory/inhibitory synaptic inputs (Liu 2004). Neuronal excitability after learning is impacted by synaptic plasticity, but also by long-term changes in neuronal properties, such as membrane potential, spike threshold, and afterhyperpolarization (Daoudal and Debanne 2003; Saar and Barkai 2003). In the hippocampus, operant conditioning training changes the intrinsic properties of CA1 neurons, without affecting the resting membrane potential or resistance (Moyer et al. 1996; Saar et al. 1998). Olfactory training reduces afterhyperpolarization to enhance excitability in the piriform cortex, suggesting long-term plasticity of intrinsic excitability (Saar and Barkai 2003). However, it is completely unknown whether motor training changes neuronal properties in the M1. Here we show that an accelerated rotor rod task promoted dynamic changes in the glutamatergic, GABAergic, and intrinsic plasticity in M1 layer II/III neurons. These findings provide functional evidence for the structural remodeling of dendritic spines after the motor skills training. Kida et al. | 3495 performed according to the Guidelines for Animal Experimentation of Yamaguchi University School of Medicine, and approved by the Institutional Animal Care and Use Committee of Yamaguchi University. Motor Learning Test To evaluate changes in motor skills, we conducted the rotor rod test (ENV577; Med Associates Inc., St. Albans, VT, USA) with 4-week-old rats (Fig. 1A). Rats were assigned to either the naive group (untrained), the training group for one day (1-day trained), or the training group for 2 successive days (2-days trained). For each test, the rats were allowed 10 attempts with a 30-s time interval between trials. But in Figure 1D, the rats were subjected additional 5 attempts after the bilateral microinjections of drugs. The interval between the 10th trial and the 11th trial was 1–2 min. The rotor rod was set to increase from 4 to 40 rpm over 5 min, and the duration of rod-riding was recorded. Microinjection of Drugs To investigate how glutamatergic neurotransmission impacted motor function, CNQX or APV was infused into M1 layers II/III. With the rats under sodium pentobarbital anesthesia (30–50 mg/kg, i.p.), a stainless-steel guide cannula (CXGW-4; Eicom Co., Kyoto, Japan) was bilaterally implanted into the M1 layer II/III region according to the brain atlas (Paxinos and Watson 1998). The coordinates were 1.2 mm anterior to bregma, 2.0 mm lateral to the midline, and 0.3 mm below the dura surface. After cannula implantation, a stylet was inserted into the guide until drug injection (Mitsushima et al. 2009, 2013). One to 3 days after cannula implantation, the stylet was replaced with an injector and the experiment was conducted. We performed bilateral microinjection of vehicle (1 µL 13% DMSO per side), CNQX (1 µg/µL per side), or APV (1 µg/µL per side). Total amount of microinjection was 2 µL/animal. One minute following completion of injections, the injector cannula was removed from the guides. To investigate the mechanism of memory acquisition, the first trial on the first day of training was initiated immediately after bilateral microinjection of vehicle, CNQX, or APV into the M1. To confirm acute effect of bilateral microinjection of CNQX on motor function, 1-day trained rats were tested in an open field test for 5 min immediately after the bilateral microinjection of vehicle or CNQX. The open field was a rectangular plastic box (length: 50 cm, width: 50 cm, height: 40 cm) with its floor equally divided into 36 squares and illuminated by white light (20 W). Sixteen squares were defined as the center, remaining 20 squares as the periphery. A rat was placed the center of the apparatus and its exploration was recorded with a video camera connected to tracer software (Muromachi Kikai Co., Tokyo, Japan). After the video recording, we analyzed time spent in the center area and total traveled distance during 5 min. Electrophysiology Materials and Methods Animals Male Sprague-Dawley rats (n = 166, Chiyoda Kaihatsu Co., Tokyo, Japan) were housed in individual plastic cages (40 × 25 × 25 cm), maintained at a constant temperature of 23 ± 1°C under a 12-h light/dark cycle, with water and food provided ad libitum. We used 67 rats for electrophysiology, 48 rats for Western blotting, and 51 rats for microinjection studies. All experiments were Thirty minutes after their final rotor rod test, the 1-day or 2-days trained rats were anesthetized with pentobarbital, acute brain slices were prepared and whole-cell recordings were performed as previously described (Mitsushima et al. 2011, 2013). Briefly, the brains were quickly perfused with ice-cold dissection buffer (25.0 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 0.5 mM CaCl2, 7.0 mM MgCl2, 25.0 mM glucose, 110.0 mM choline chloride, and 11.6 mM ascorbic acid) and gassed with 5% CO2/95% O2. Coronal brain slices were cut into 350-μm slices with a Leica 3496 | Cerebral Cortex , 2016, Vol. 26, No. 8 Figure 1. (A) Experimental design of the rotor rod task. (B) Mean latency to fall from the barrel plotted for each trial (10 trials/day) in 2-days trained rats. (C) Mean latency at the first trial and the final trial on the first and second days of training. **P < 0.01 versus first. (D) Vehicle (open), CNQX (gray), or APV (filled) was bilaterally injected into the M1 after the 10th trial. *P < 0.05 versus vehicle. (E) Visualization of the microinjected site in the M1. CC, corpus callosum; M2, secondary motor cortex. (F) Hematoxylin staining of injection site (black asterisk). Scale bars, 200 µm. (G) Open field performance immediately after the bilateral microinjection of CNQX. (H) Vehicle, CNQX, or APV was bilaterally microinjected into the M1 before the 1st trial. Arrows indicate the microinjection timing. The number of rats in each group is shown in parentheses or at the bottom of each bar. *P < 0.05, **P < 0.01 versus vehicle. Error bars indicate ± SEM. Motor Training-Dependent Neocortical Plasticity vibratome (VT-1200; Leica Biosystems, Nussloch, Germany) in dissection buffer and transferred to physiological solution (22– 25°C, 118 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 10 mM glucose, 4 mM MgCl2, and 4 mM CaCl2, pH 7.4, gassed with 5% CO2/95% O2). We maintained 3–4 slices from each rat, and then selected 1–2 brain slices for patch recordings based on the brain atlas by Paxinos and Watson (1998). Glass electrodes were made with a horizontal puller (Model P97; Sutter Instrument, Novato, CA, USA) and filled with a suitable solution. Whole-cell recordings were obtained from pyramidal neurons of M1 layer II/III, using an Axopatch-1D amplifier (Axon Instruments Inc., Union City, CA, USA). Recordings were digitized using a Digidata 1440 AD board (Axon), recorded at 5 kHz, and analyzed offline with pCLAMP 10 software (Axon). The coordinates of recording cells were ∼1.2 mm anterior to bregma, 2.0 mm lateral to the midline, and 0.2–0.3 mm below the dura surface: the region corresponding to M1 forelimb representation (Neafsey et al. 1986; Kleim et al. 2002). Voltage Clamp Recordings The recording chamber was perfused with 22–25°C physiological solution. For the analysis of AMPA/NMDA ratio, we added 0.1 mM picrotoxin to block GABAA receptor-mediated inhibition, and 4 μM 2-chloroadenosine to stabilize evoked neuronal responses (Baidan et al. 1995). Patch recording pipettes (4–7 MΩ) were filled with intracellular solution (115 mM cesium methanesulfonate, 20 mM CsCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM sodium phosphocreatine, and 0.6 mM EGTA at pH 7.25). A bipolar tungsten stimulating electrode (Unique Medical Co., Ltd., Tokyo, Japan) was positioned in the M1 region at 200–300 µm lateral from the recorded neurons. Stimulus intensity was increased from 0.4 to 0.7 mA until a synaptic response with amplitude of >10 pA was recorded. The AMPA/NMDA ratio was calculated as the ratio of the peak current at −60 mV to the current at + 40 mV at 150 ms after stimulus onset (50–100 traces were averaged for each holding potential). Miniature Recordings For miniature recordings, we used modified intracellular solution to adjust the reversal potential of the GABAA receptor response (127.5 mM cesium methanesulfonate, 7.5 mM CsCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM sodium phosphocreatine, 0.6 mM EGTA, pH 7.25). Moreover, we added 0.5 µM tetrodotoxin (TTX; Wako Pure Chemical Industries Ltd., Osaka, Japan) and 0.1 mM APV (Sigma-Aldrich Co., Tokyo, Japan) to perfusate to block both action potentials and NMDA receptor-mediated excitatory postsynaptic currents. The voltage was clamped at −60 mV for mEPSC recording, and at +15 mV for mIPSC recording. We analyzed the frequency and amplitude of mEPSCs/mIPSCs above 10 pA. To calculate excitation and inhibition (E/I) balance, the value of miniature excitatory postsynaptic current (mEPSC) frequency or amplitude was divided by corresponding value of miniature inhibitory postsynaptic current (mIPSC) frequency or amplitude in each neuron. After recording, we confirmed that mEPSCs and mIPSCs were completely abolished by 10 µM CNQX (Sigma) and 10 µM bicuculline methiodide (Sigma), respectively. Paired-Pulse Stimulation To analyze presynaptic plasticity at excitatory synapses, we added 0.1 mM picrotoxin and 4 μM 2-chloroadenosine to the perfusate and performed paired-pulse stimulation at −60 mV. To analyze presynaptic plasticity at inhibitory synapses, we added Kida et al. | 3497 0.1 mM APV and 4 μM 2-chloroadenosine to the perfusate and performed paired-pulse stimulation at +15 mV. To evaluate the paired-pulse ratio from the EPSC or IPSC average, 50–100 sweeps were recorded with paired stimuli at 100-ms intervals. The ratio of the second amplitude to the first amplitude was calculated as the paired-pulse ratio. Current Clamp Recordings For current clamp recordings, pipettes were filled with 130 mM K-Gluconate, 5 mM KCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM Na-phosphocreatine, and 0.6 mM EGTA at pH 7.25. Liquid junction potential was not corrected. During 300-ms current injections from −100 to +400 pA, we counted the numbers of spikes in neurons. To determine the threshold for each neuron, we identified the minimal voltage for action potential induction. The amplitude of afterhyperpolarization was evaluated by measuring the voltage at spike initiation and the lowest voltage during AHP (van der Velden et al. 2012). To further examine the role of AMPA and NMDA receptors on changes of intrinsic properties, CNQX (1 µg/µL) or APV (1 µg/µL) was unilaterally microinjected into the M1 prior to the motor training. After the 10 sessions of training on day 1, we made acute brain slices of drug-injected side for the current clamp analyses (Fig. 7) Western Blotting Western blotting was performed according to the previous study (Yamamoto et al. 2013). Thirty minutes after training, the brain was removed and incubated for 3 min in ice-cold buffer containing 0.32 M sucrose and 20 mM Tris–HCl (pH 7.5). Dissected motor cortical tissues were homogenized in 200 µL of buffer containing 50 mM Tris–HCl ( pH 7.4), 0.5% Triton X-100, 0.5 M NaCl, 10 mM EDTA, 4 mM EGTA, 1 mM Na3VO4, 50 mM NaF, 40 mM sodium pyrophosphate, 1 mM protease inhibitor, and 1 mM DTT. Insoluble material was removed by a 10-min centrifugation at 15,000 rpm. For the analysis of synaptosomal fraction, we used the SynPER Reagent (Thermo Scientific) with protease/phosphatase inhibitor cocktail (100×). Briefly, samples were centrifuged at 1200 G for 10 min, and the remaining supernatant centrifuged at 15 000 G for 20 min to obtain synaptosome pellet. The resulting pellet was resuspended in 50 μL of lysis buffer. Samples containing equivalent amounts of protein based on the bicinchoninic acid analysis (Thermo Scientific, Rockford, IL, USA) were heated at 100°C for 3 min in Laemmli sample buffer and subjected to SDS-PAGE for 30 min at 200 V. Proteins were transferred to an Immobilon PVDF membrane for 1 h at 100 V. Membranes were blocked for 1 h at room temperature in T-TBS solution containing 50 mM Tris–HCl ( pH 7.5), 150 mM NaCl, 0.1% Tween 20, and 5% skim milk, followed by overnight incubation at 4°C with anti-GluA1 (1:1000; Millipore, Tokyo, Japan), anti-phospho-GluA1 (Ser831) (1:1000; Millipore), and anti-β-Tubulin (1:1000; BioLegend, San Diego, CA, USA). This step was followed by incubation with HRPconjugated goat anti-rabbit IgG for phospho-GluA1 (1:5000, Millipore) and HRP-conjugated goat anti-mouse IgG (1:5000, Millipore) for GluA1 and β-Tubulin. Bound antibodies were visualized using an enhanced chemiluminescence detection system (GE Healthcare, Chalfont St. Giles, UK) and semiquantitatively analyzed using the Image-J program (National Institutes of Health, Bethesda, MD, USA). Histology To confirm the injected sites, we further microinjected a retrograde tracer after the behavioral experiment (Fig. 1E, LumaFluor 3498 | Cerebral Cortex , 2016, Vol. 26, No. 8 Inc., Durham, NC, USA). The animals were deeply anesthetized and perfused with a 4% paraformaldehyde solution. Frozen coronal sections (50-µm thick) were sequentially cut using a microtome. The location of the microinjected site was microscopically verified in the frozen sections. Some rats were used for hematoxylin staining without the tracer microinjection (Fig. 1F). The animals were deeply anesthetized and perfused transcardially with phosphate-buffered saline, followed by 4% paraformaldehyde in phosphate buffer. The brain was removed and post-fixed in 4% paraformaldehyde for 4–12 h. The brain was embedded in paraffin, and then cut into coronal sections (4-μm thick) with the microtome. Sections were affixed to silane-coated glass slides for staining. Data Analysis To determine the development of motor skill, the data on the latency to fall were analyzed by one-way analysis of variance (ANOVA) with repeated-measures followed by post hoc analysis with the Fisher’s protected least significant difference (PLSD) test, where variable was trial. To compare the latency to fall between the drug-injected groups, we used 2-way ANOVA with repeated-measures, followed by post hoc ANOVAs with the Fisher’s PLSD test. To compare the differences between 2 groups in open field study, we used unpaired 2-tailed t-test. To compare the means of multiple individual groups, we used one-way factorial ANOVA followed by post hoc analysis with Fisher’s PLSD test. E/I balance was analyzed using the Kruskal– Wallis test followed by post hoc analysis with Scheffe’s test. To evaluate correlations between mEPSC and mIPSC parameters, we calculated Spearman’s correlation coefficient. The current injection data were analyzed by 2-way ANOVA with repeated measures followed by post hoc ANOVAs with the Fisher’s PLSD test. P values of