在P0在新生小鼠解剖示蹤研究表明，皮質(zhì)脊髓運(yùn)動神經(jīng)元位于在運(yùn)動皮層V層已發(fā)出的軸突脊髓（阿羅塔等人，2005；莫利諾等人，2007）。主軸陣陣已發(fā)現(xiàn)的爪子S1活動的唯一模式（khazipov等人，2004）。然而，什么樣的活動模式表示在M1和自發(fā)的運(yùn)動，他們的關(guān)系在早期發(fā)育階段是未知的。還不清楚什么S1和M1和M1 S1神經(jīng)活動的功能角色之間的相互作用。為了解決這些問題，我們同時進(jìn)行多通道細(xì)胞外電生理學(xué)記錄在S1和M1，通過電壓敏感染料反應(yīng)爪子觸摸刺激后的功能鑒定。
 

1聲明
本文包含了一些零件，已發(fā)表或?qū)l(fā)表在體內(nèi)的新生大鼠桶狀皮層長時程增強(qiáng)，γ和新生大鼠皮層驅(qū)動主軸陣陣，早期活動觸發(fā)。

2材料和方法
項目1
所有的實(shí)驗(yàn)都進(jìn)行按照研究中使用的動物的國家法律，由當(dāng)?shù)氐膫惱砦瘑T會批準(zhǔn)。電壓敏感染料成像（VSDI），場電位（FP）和多單位活動（MUA）記錄在頭部約束新生Wistar大鼠P0～P14桶皮質(zhì)。
 

3.手術(shù)準(zhǔn)備

 

外科手術(shù)是根據(jù)所描述的方法進(jìn)行（漢嘉努等人。，2006；漢嘉努等人。，2007；楊等人。，2009）。把深冰冷卻單獨(dú)麻醉下注入光是P0，P7大鼠腹腔注射（0.5克/公斤，西格瑪奧德里奇，Taufkirchen，德國）。技能，在頭骨的軟組織被小心地取出。然后，暴露顱骨是干的（圖11）。接下來，桶狀皮層標(biāo)有紅色（桶皮質(zhì)，p0-p1,0-2毫米后方囟和2-4毫米從中線；p3-p5,0-2毫米后方囟，2.5-4.5毫米從中線；p6-p7,1-3毫米后方囟，3-5毫米從中線）。在那之后，頭被固定在立體定位儀上用一架固定牙科水泥枕骨。骨，但不是硬腦膜，S1是鉆一個2×2平方毫米開顱仔細(xì)去除（圖12A1）。后來，動物的身體被包圍的棉花和被放置在一個加熱毯保持在一個恒定的37攝氏度溫度。
 

Anatomical tracer studies in newborn mice at P0 have demonstrated that, corticospinal motor neurons located at layer V in motor cortex have already sent the axons to spinal cords (Arlotta et al., 2005;Molyneaux et al., 2007). Spindle bursts have been found to be the only patterns of activity in paws S1 (Khazipov et al., 2004). However, what the patterns of activity expressed in M1 and their relation to spontaneous movements during the early developmental stages are unknown. It is also not clear what the interactions between S1 and M1 and what the functional roles of S1 neuronal activity in M1. To address these questions, we simultaneously performed multichannel extracellular electrophysiological recordings in S1 and M1 that were functionally identified by voltage-sensitive dye responses following paw touch stimulation.
 

1Statement
This thesis contains some parts, which have already been published or will be published: Long-term potentiation in the neonatal rat barrel cortex in vivo (An et al., 2012). Gamma and spindle bursts in neonatal rat motor cortex drive and are triggered by early motor activity (An et al. in preparation).#p#分頁標(biāo)題#e#

2Materials and methods
Project 1
All experiments were carried out in accordance with the national laws for the use of animals in research and approved by the local ethical committee . Voltage-sensitive dye imaging (VSDI), field potential (FP) and multiple-unit activity (MUA) recordings were made in the barrel cortex of head restrained neonatal Wistar rats P0 to P14.

3.Surgical preparation
The surgical procedure was performed according to the methods as described previously (Hanganu et al., 2006;Hanganu et al., 2007;Yang et al., 2009). P0 to P7 rats were put under deep ice-cooling anesthesia alone and injected with light intraperitoneal (0.5-1 g/kg, Sigma-Aldrich, Taufkirchen, Germany). The skill and the soft tissue over the skull were carefully removed. Then, the exposed skull was dry (Fig. 11). Next, barrel cortex is marked with the red dots (barrel cortex, P0-P1,0-2 mm posterior to bregma and 2-4 mm from the midline; P3-P5,0-2 mm posterior to bregma and 2.5-4.5 mm from the midline; P6-P7,1-3 mm posterior to bregma and 3-5 mm from the midline). After that, the head was fixed into the stereotaxic apparatus using one holder fixed with dental cement occipital bones. The bone, but not the dura mater, over S1 was carefully removed by drilling a 2 x 2 mm2 craniotomy (Fig 12A1). Afterwards, the body of the animals was surrounded by cotton and kept at a constant temperature of 37? C by placing it on a heating blanket. During recordings, milk was fed to reduce the distress when the pups showed any sign of thirsty.

HEPES (pH 7.3 with NaOH). The voltage-sensitive dye was topically applied to the surface of the barrel cortex and allowed to diffuse into the cortex for 15 to 30 min (Fig 12A2). Subsequently, unbound dye was carefully washed away with saline solution. This procedure resulted in a complete staining of all cortical layers from the subplate to the marginal zone / layer I in P0-P1 rats and in a more superficial staining pattern in P6-P7 animals , similar as described previously for adult rodent cerebral cortex (Berger et al., 2007;Ferezou et al., 2007). The cortex was covered with 1% low-melting agarose and a cover slip was placed on top to stabilize the tissue. Excitation light from a red LED (MRLED 625 nm, Thorlabs GmbH, Dachau, Germany) was band pass filtered (630/30 nm) and reflected towards the sample by a 650 nm dichroic mirror (Fig 12A3, 4). The excitation light was focused onto the cortical surface with a 25 mm Navitar video lens (Stemmer Imaging, Puchheim, Germany). Emitted fluorescence was collected via the same optical pathway, but without reflection of the dichroic mirror, long pass filtered (660 nm) and focused via another 25 mm Navitar lens onto the chip of a MiCam Ultima L high speed camera (Scimedia, Costa Mesa, CA, USA). This tandem-type macroscope design (Ratzlaff and Grinvald, 1991) resulted in a 1x magnification. As the high speed camera has a detector with 100x100 pixels and a chip size of 10x10 mm2, the field of view was 10x10 mm2. Using a C-mount extension tube we reduced the field of view to 2.6x2.6 mm2 and thereby reduced in addition the vignetting. Every pixel collected light from a cortical region of 26x26 μm2 (Fig. 12B1). The tandem-type macroscope comprising the LED, the filter cube and the two video optics were built in the mechanical workshop of our institute. Fluorescence measurements were synchronized to electrophysiological recordings through TTL pulses.#p#分頁標(biāo)題#e#
Spontaneously ongoing activity was detected during 16 s long imaging sessions while evoked activity following whisker stimulation was imaged in 2 s long sessions, both with an unbinned whole frame sampling frequency of 500 Hz. For both sets of experiments, data were not averaged.

After VSDI recording, VSDI evoked response image (Fig. 12B2) and photo of craniotomy with clear blood vessel (Fig. 12B3) merged together, which can provide the precise position for multi-channel recording.

4Evaluation of voltage-sensitive dye imaging data
電壓敏感染料成像數(shù)據(jù)
Images were analyzed offline using custom-made routines in MATLAB software version 7.7 (Mathworks, Natick, MA). In order to improve the signal to noise ratio, the image data were first processed using 5x5 pixel spatial binning followed by 60 Hz low pass filtering. Bleaching of fluorescence was corrected by subtraction of a best-fit double-exponential or 5th degree polynomial (curve fitting tool in MATLAB). The normalized change of fluorescence intensity(△F / F0 ) was calculated as the change of fluorescence intensity (△F) in each pixel divided by the initial fluorescence intensity (F0) in the same pixel.
Only fluorescent changes with a maximal △F / F0 of at least 0.2% were considered as evoked responses or spontaneous events. To evaluate their onset, 3x3 pixels were taken around a pixel with a signal at least 7 times higher than the baseline standard deviation, which represented the point of earliest activity as determined by the experimenter. The duration of the events was determined as the time in which the signal was above the half-maximal △F / F0 amplitude. The area of the evoked response or spontaneous event was defined as the contour plot of the VSDI response with reference to the half-△F / F0 amplitude. The spatial representation of VSDI responses were displayed according to this threshold. The diameter of the VSDI responses were calculated from the area of VSDI responses under the assumption of a circular response (d=2(a/ π)1/2).

5 Multi-electrode recording protocols
多電極記錄報告
Protocols were similar to those described previously (Yang et al., 2009). According the positions of blood vessel and central positions of principle barrels in the last merged picture (Fig. 12B3), we could perform multi-channel recording. Field potential (FP) and multiple unit activity (MUA) were recorded by a four-shank (Fig. 17A1) or a one-shank (Fig. 20A) 16-channel Michigan electrode (1-2 MΩ, NeuroNexus Technologies, Ann Arbor, MI) into one barrel column according to VSD response. The recording sites were separated by 125 μm in horizontal direction and 50 in vertical direction for the four-shank electrode (Fig. 17A1). Moreover, the recording sites in one-shank electrode were separated by 100μm at P3-P5 (Fig. 20B) and 50 μm at P0-P1 (Fig. 20C). Both two kinds of electrodes were labelled with DiI (1,1’-dioctadecyl-3,3,3’,3’-tetramethyl indocarbocyanine, Molecular Probes, Eugene, OR, USA). Dil stained tracks could help to reconstruct electrodes array position on Nissl-stained coronal section (Fig.20A). FP and MUA were recorded at least for 2 hours at a sampling rate of 20 kHz using a multi-channel extracellular amplifier and the MC_RACK software (Multi Channel Systems).#p#分頁標(biāo)題#e#

6 Calculate the slope of FP
FP邊坡計算
The efficacy of synaptic transmission was calculated from the slope of the initial negativation of the evoked FP response (Fig. 13A). Evoked FP responses were visually inspected to exclude artifacts or contaminations with spontaneous activity (Fig. 13B, C). The slope was determined between 20 and 80% of maximal FP amplitude and was normalized to the average slope recorded during a 30 min baseline interval. The largest FP slopes located in layer IV at P3-P7 and cortical plate at P0-P1 were chosen for comparing among different ages (Fig. 15B), spatial locations (Fig. 17-18) and layers groups (also in layer II/III at P3-P5 and deep layer at P0-P1, Fig. 21). For statistical analyses slope values were averaged for a 5-30 min and 35-60 min poststimulus intervals. Data analysis was performed with MATLAB software versions R2008B. ? ? ?

One way ANOVA test which followed by multiple comparisons with Bonferroni correction was performed to compare more than two groups at 35-60 min after 2 Hz stimulation.

surface close to the multi-channel recording electrode. After a 30 min baseline recording, 2–4 μl lidocaine was applied. Blockade of M1 or S1 lasted approximately for 30 min.
Local inactivation of neuronal activity from the forepaw to M1 was achieved by application of lidocaine (3% in saline). After a 30 min baseline recording, 40 μl of lidocaine was injected into forepaw. Blockade of M1 or S1 lasted approximately for 1 hour.
3.2.8 Analysis of multi-electrode recordings data
As described previously (Yang et al., 2012), the FP signals from each channel were analyzed using the unfiltered data. MUA was detected using 200 Hz high-pass filtered signals with a threshold at 5 times the baseline SD and a bin of 1 ms. The post-stimulus time histograms (PSTH) were analyzed by summing up the activity in ~20 trials and normalized to number of spikes per second per trial. Sliding window method with 10 ms window and 1 ms step was applied for the PSTH calculation.
Gamma and spindle bursts were detected as follows. FPs contained at least 3 cycles with a duration less than 120 ms (the frequency more than 25 Hz), we defined them as gamma busts.. Similarly, FPs contained at least 3 cycles with a duration more than 150 ms (the frequency less than 20 Hz), we defined them as spindle busts. Furthermore, both gamma and spindle bursts should be accompanied by MUA.
The time–frequency spectrogram, power spectra, and coherence were analyzed using unfiltered raw data. Matlab spectrogram function with a time window of 100 ms and an overlapping of 99 ms (Matlab 7.7, Math works) was used for the time–frequency spectrogram analyses. Chronux toolbox  was used for spectrum and coherence analyses. Furthermore, Jackknife method provided in the Chronux toolbox was used to calculate 95% confidence intervals (CIs). Both spectrum and coherence analyses were performed using a time–bandwidth product of TW= 1 with K = 1 taper, and the padding factor for the fast fourier transformation (FFT) was 2.#p#分頁標(biāo)題#e#

7 Statistical tests統(tǒng)計實(shí)驗(yàn)
Data are presented as mean ± s.e.m. Statistics were tested with paired t-test (for comparing subsequent measurements in the same group of animals) and one way ANOVA (for comparing more than two different groups) tests followed by multiple comparisons with Bonferroni correction using SPSS software version 13.0 or Mann–Whitney–Wilcoxon test for the data sets recording in two sites but from one animal using GraphPad Prism (GraphPad Software, Inc.).

 Results結(jié)果
1 Project 1
1.1min whisker stimulation at 2 Hz induces age-dependent expression of LTP in the neonatal rat barrel cortex
The FP responses to single whisker stimulation recorded in newborn rat barrel cortex in vivo consisted of an early gamma activity followed by spindle bursts as described previously (Minlebaev et al., 2007;Yang et al., 2009;Minlebaev et al., 2011;Yang et al., 2012). To analyze activity-dependent modifications of the evoked responses, we quantified the slope of the initial negative going FP response, which reflects the early activation of the cortex via the thalamocortical pathway.
In P3-P5 rats (n=16 pups) repetitive single whisker stimulation at 2 Hz for 10 min (Fig. 14) induced a significant (F(1.44,21.58)=0.77, p<0.001) increase in the slope of the FP that persisted for >60 min. The FP slope increased dramatically (P<0.001, P<0.001 for 5-30 min, 35-60min against baseline, respectively) to 183.7 ± 12.4% (n=16) during the 5-30 min interval and to 208.1 ± 14.0% during the 35-60 min interval after LTP induction (upper traces in Fig. 15A1, red symbols in Fig. 15A2, 3). No significant changes (F(2,16)=1.66, P=0.22) in the FP slope (97.60 ± 2.39, 100.66 ± 2.83, 105.57 ± 4.64 at baseline, 5-30 min, 35-60 min, respectively) could be observed in the age-matched control group (n=9 pups), which did not receive repetitive 2 Hz whisker stimulations (lower traces in Fig. 15A1, open squares in Fig. 15A2, 3). Moreover, the relative FP slope showed the larger potentiation during the post stimulation recording than control group (P<0.001, P<0.001 for 5-30 min, 35-60 min in the stimulation group against control group respectively). These data demonstrate for the first time that the cerebral cortex of P3-P5 rats shows a prominent LTP to physiological stimulation of the afferent pathway in vivo.
Previous in vitro studies have documented in thalamocortical slices of newborn rats that the magnitude of LTP in barrel cortex gradually decreases between P3 and P7and LTP cannot be induced after the first postnatal week (Crair and Malenka, 1995). In order to address the question whether a similar age-dependent expression of LTP can be also observed in vivo to physiologically relevant afferent stimulation, we studied the expression of LTP in P0-P1 and P6-P14 rats. In P0-P1 animals (n=12), single whisker stimulation at 2 Hz for 10 min induced a significant (F(2,22)=79.30,p<0.001) and stable LTP (P<0.001, P<0.001 for 5-30 min, 35-60min against baseline, respectively ) during the 5-30 min (161.7 ± 6.1%) and 35-60 min (150.7 ± 3.6%) post-induction interval (filled black squares in Fig. 15B1, B2). In P6-P7 rats (n=10), single whisker stimulation elicited an increase in the FP slope during the 5-30 min (146.5 ± 19.5%) and 35-60 min (149.9 ± 23.6%) post-induction interval, which were, however, not significantly (F(1.16,10.43)=4.39, p=0.351) different from the baseline responses (blue symbols in Fig. 15B1, B2). In P8-P14 (n=5) rats no obvious changes in the FP slope (98.68 ± 1.1, 99.30 ± 3.9, 105.3 ± 5.6 at baseline, 5-30 min, 35-60 min, respectively; F(2,8)=0.98, P=0.417) could be evoked (green symbols in Fig. 15B1, B2). Furthermore, the 35-60 min LTP phase was significantly (p=0.027, P=0.037, for P3-P5 against P0-P1, P6-P7 respectively) smaller in P0-P1 and P6-P7 animals when compared to the P3-P5 group. In summary, these results indicate that LTP is limited to the critical periods with highest magnitude at P3-P5.#p#分頁標(biāo)題#e#

2 Schematic diagram of the experimental setup.
實(shí)驗(yàn)裝置示意圖
Schematic diagram of the experimental setup illustrating selective mechanical stimulation of the C2 whisker (A) and simultaneous VSDI in the barrel cortex (B). The exposed barrel cortex was stained with the voltage sensitive dye RH1691. A single whisker deflection of the C2 whisker elicits a local VSDI response in a P3 rat. The green dot indicates the centre of the C2 barrel-related cortical column. The black dot is the electrode insertion position. The orange color represent the region of the C2 whisker stimulation evoked VSDI response. C. shows the same area after termination of the electrophysiological recording and retraction of the recording electrode. The red dot shows the electrode insertion point (indicated by arrowhead). D. Stimulation protocol for induction of LTP. During baseline recording, the whisker was deflected twice per 5 min at 1 min interval for 30 min. For LTP induction the whisker was deflected at a frequency of 2 Hz for 10 min. Afterwards the same 2 stimuli per 5 min were used again for 60 min during the post stimulation recording period

3 Mechanical deflection of a single whisker for 10 min at 2 Hz elicits LTP of FP slope in barrel cortex of newborn rats in vivo.
在2 Hz 10分鐘的一個單一的晶須機(jī)械偏轉(zhuǎn)誘發(fā)LTP的新生大鼠桶狀皮層FP坡體內(nèi)。
A.Time course of FP responses before and after induction of LTP. A1. Representative FP recording during baseline, the 5-30 min and 35-60 min phases after 2 Hz stimulation (red) or without 2 Hz stimulation (black) in a P4 rat. A2. Relative FP slopes recorded in P3-P5 control and LTP group. Data are expressed as mean ± s.e.m. A3. Box plots of FP slopes in P3-P5 control and LTP group with baseline, 5-30 min and 35-60 min post stimulation. B1. Relative FP slopes recorded in different age groups. Data are expressed as mean ± s.e.m. B2. Box plots of FP slopes in LTP groups of different ages with baseline, 5-30 min and 35-60 min post stimulation. Significant levels between different intervals were tested with paired t-test, differences to control experiments were tested with Mann–Whitney–Wilcoxon test, and differences among age groups were tested with one-way ANOVA followed by multiple comparisons. Significance levels of p<0.001 (***) and p<0.05 (*) were identified.

Furthermore, we quantified the spike number of single whisker evoked response to analyze activity-dependent modifications, which reflects the early plasticity of the cortex via the neuronal network.
Similarly, in P3-P5 rats (n=16 pups) repetitive single whisker stimulation at 2 Hz for 10 min induced a significant (F(2,30)=54.547, p<0.001) increase in the spike number of single evoked response that persisted for >60 min. The the spike number increased dramatically (P<0.001, P<0.001 for 5-30 min, 35-60min against baseline, respectively) to 163.5 ± 9.0% (n=16) during the 5-30 min interval and to 194.0 ± 9.7% during the 35-60 min interval after LTP induction (upper traces in Fig. 16A1, red symbols in Fig. 16A2, 3). No significant changes (F(1.3,10.1)=2.373, P=0.152) in the spike number (101.7 ± 1.1, 107.0 ± 2.0, 109.6 ± 5.2 at baseline, 5-30 min, 35-60 min, respectively) could be observed in the age-matched control group (n=9 pups), which did not receive repetitive 2 Hz whisker stimulations (lower traces in Fig. 16A1, open squares in Fig. 16A2, 3). Moreover, the relative the spike number showed the larger potentiation during the post stimulation recording than control group (P<0.001, P<0.001 for 5-30 min, 35-60 min in the stimulation group against control group respectively). Again, these data also demonstrate for the first time that the cerebral cortex of P3-P5 rats shows a prominent LTP to physiological stimulation of the afferent pathway in vivo.

Similary, we studied the MUA acitivity of LTP in P0-P1 and P6-P14 rats. In P0-P1 animals (n=12), single whisker stimulation at 2 Hz for 10 min induced a significant (F(2,22)=35.4, p<0.001) and stable LTP (P<0.001, P<0.001 for 5-30 min, 35-60min against baseline, respectively ) during the 5-30 min (139.7 ± 6.5%) and 35-60 min (149.2 ± 5.6%) post-induction interval (filled black squares in Fig. 16B1, B2). In P6-P7 rats (n=10), single whisker stimulation elicited an increase in the spike number during the 5-30 min (124.6 ± 8.9%) and 35-60 min (137.1 ± 11.8%) post-induction interval, which were significantly (F(2,18)=7.8, p=0.004) increase than the baseline responses (blue symbols in Fig. 16B1, B2). In P8-P14 (n=5) rats no obvious changes in the spike number (99.3 ± 2.3, 103.5 ± 4.6, 97.4 ± 1.2 at baseline, 5-30 min, 35-60 min, respectively; F(2,8)=0.8, P=0.474) could be evoked (green symbols in Fig. 16B1, B2). Furthermore, the 35-60 min LTP phase was significantly(p=0.004, P<0.001, for P3-P5 against P0-P1, P6-P7 respectively) smaller in P0-P1 and P6-P7 animals when compared to the P3-P5 group. In summary, these results consistent with FP slope of LTP and again indicate that LTP is limited to the critical periods with highest magnitude at P3-P5.

Fig. 16 Mechanical deflection of a single whisker for 10 min at 2 Hz elicits LTP of MUA in barrel cortex of newborn rats in vivo.
A.Time course of MUA samples before and after induction of LTP. A1. Representative MUA recording during baseline, the 5-30 min and 35-60 min phases after 2 Hz stimulation (red) or without 2 Hz stimulation (black) in a P4 rat. A2. Relative spike number recorded in P3-P5 control and LTP group. Data are expressed as mean ± s.e.m. A3. Box plots of spike number in P3-P5 control and LTP group with baseline, 5-30 min and 35-60 min post stimulation.B1. Relative spike number recorded in different age groups. Data are expressed as mean ± s.e.m. B2. Box plots of spike number in LTP groups of different ages with baseline, 5-30 min and 35-60 min post stimulation. Significant levels between different intervals were tested with paired t-test, differences to control experiments were tested with Mann–Whitney–Wilcoxon test, and differences among age groups during 35-60 min phase were tested with one-way ANOVA followed by multiple comparisons. Significance levels of p<0.001 (***), p<0.01 (**) and p<0.05 (*) were identified.

35-60 min intervals, respectively; n= 14 channels in 12 pups, Fig. 17C1, C2). A lower LTP expression was observed at distances between 100 and 200 μm (125.9 ± 8.6% and 134.8 ± 6.2% respectively, n= 8 channels in 4 pups), while no significant LTP could be observed at electrode distances >200 μm (n= 7 channels in 4 pups, Fig. 17C1, C2). These suggest that the expression of LTP in newborn rat barrel cortex is mostly restricted to the activated barrel-related column.

same distance groups , ***, p < 0.001, **, p < 0.01, *, p < 0.05 for 5-30 and 35-60 min LTP phases against baseline respectively, repeated measures ANOVA followed by multiple comparisons with Bonferroni correction. In three different distances, , p < 0.001, , p < 0.01， , p<0.05, for relative FP slope in < 100μm against 200 - 300μm,100 - 200μm during 35-60 min phase respectively, one-way ANOVA followed by multiple comparisons with Bonferroni correction.