血管鋳造および組織透明化による脳血管網の観察

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 池谷 裕二, 東京大学教授 浦野 泰照, 東京大学講師 岸 雄介, 東京大学講師 前田 和哉, 東京大学講師 本間 雅University of Tokyo(東京大学

Dopamine receptor activation reorganizes neuronal ensembles during hippocampal sharp waves in vitro.

Hippocampal sharp wave (SW)/ripple complexes are thought to contribute to memory consolidation. Previous studies suggest that behavioral rewards facilitate SW occurrence in vivo. However, little is known about the precise mechanism underlying this enhancement. Here, we examined the effect of dopaminergic neuromodulation on spontaneously occurring SWs in acute hippocampal slices. Local field potentials were recorded from the CA1 region. A brief (1 min) treatment with dopamine led to a persistent increase in the event frequency and the magnitude of SWs. This effect lasted at least for our recording period of 45 min and did not occur in the presence of a dopamine D1/D5 receptor antagonist. Functional multineuron calcium imaging revealed that dopamine-induced SW augmentation was associated with an enriched repertoire of the firing patterns in SW events, whereas the overall tendency of individual neurons to participate in SWs and the mean number of cells participating in a single SW were maintained. Therefore, dopaminergic activation is likely to reorganize cell assemblies during SWs

SKF38393 increases the repertoires of SW-relevant LFP spectra patterns.

<p><b>A.</b> Representative fast Fourier transform spectra of 136 SW events during the pre-treatment baseline period of 3 min. <b>B.</b> The correlation matrix obtained from the 136 SWs was sorted by the affinity propagation and was clustered into 14 SW subgroups. <b>C.</b> The mean numbers of the SW subgroups before and after administration with control aCSF (left) and SKF38393 (right). Each gray dataset indicates a single slice. **<i>P</i> = 0.0059, <i>t</i>₇ = 3.38, paired <i>t</i>-test. Data are the means ± SEMs of 8 slices.</p

D₁/D₅ receptor activation is responsible for dopamine induced SW facilitation.

<p><b>A.</b> Time course of the percentage of the frequency of SW events relative to the mean value during the pre-application period while the slices were perfused with 0.1 µM SCH23390, a D₁/D₅ receptor antagonist, and 1 µM sulpiride, a D₂ receptor antagonist, from time 0–45 min. <b>B–C.</b> The mean SW event frequency (B) and the mean SW amplitude (C) at time 30–35 min. <b>D.</b> Slices were perfused with 1 µM dopamine for 1 min at time 0 in the continuous presence of 0.1 µM SCH23390 or 1 µM sulpiride. <b>E–F.</b> The mean SW event frequency (E) and the mean SW amplitude (F) at time 30–35 min. *<i>P</i> = 0.032, <i>t</i>₆ = 2.78, *<i>P</i> = 0.035, <i>t</i>₆ = 2.71, paired <i>t</i>-test <i>versus</i> the −5-to-0 min period. Data are the means ± SEMs of 7 and 6 slices from 4 and 3 mice, respectively. <b>G.</b> Slices were perfused with 30 µM SKF38393 at time 0 in the absence (orange) and the continuous presence (light blue) of 0.1 µM SCH23390, a D₁/D₅ receptor antagonist. <b>H–I.</b> The mean SW event frequency (H) and the mean SW amplitude (I) at time 30–35 min. **<i>P</i> = 0.0017, <i>t</i>₁₂ = 4.02 (H); *<i>P</i> = 0.017, <i>t</i>₁₂ = 2.78 (I), paired <i>t</i>-test <i>versus</i> the −5-to-0 min period. Data are the means ± SEMs of 13 and 5 slices from 10 and 3 mice, respectively. <b>J.</b> While fEPSPs evoked by field stimulation of Schaffer collaterals were recorded from CA1 stratum radiatum, slices were perfused with 30 µM SKF38393 for 1 min at time 0. Time changes in fEPSP amplitudes (left) and slopes (right) are plotted as mean ± SEMs of 3 slices from 3 mice. The insets indicate example traces at two time points.</p

Neuronal activities during SWs are optically imaged.

<p><b>A.</b> A confocal image of the CA1 stratum pyramidale in an OGB1-loaded slice. The location of an LFP electrode is shown by the white lines. <b>B.</b> Example of calcium transients from 9 cells and LFP trace, before (left) and after (right) SKF38393 application. <b>C.</b> Simultaneous cell-attached recording and calcium imaging. Numbers of spikes are represented above each spike. Spike timimgs detected from the calcium trace are shown by bars below the trace. <b>D.</b> A peri-SW time histogram of calcium events. Data are the means ± SEMs from 8 slices, including a total of 7,709 calcium events emitted by 199 cells. The time period between −200 ms and 50 ms relative to the SW peak was defined as a SW window (shadow). Calcium events during this time window are regarded as SW-locked activities. <b>E.</b> Representative raster plots of calcium events 0–3 min before (top) and 15–18 min after (bottom) the bath application of 30 µM SKF38393. Green or orange bars above each raster plot indicate the time stamps of individual SW events. Thick dots in the rastergram indicate SW-locked activities. <b>F–H.</b> Comparisons of peri-SW time histograms from each slices in 3 parameters; Time lag of the histogram peak from the SW peak (F), skewness of the histograms (G) and kurtosis of the histograms (H). <b>I.</b> Comparisons of the co-activation probability of any given pairs of cells that participated at least once in SW before (left) and after (right) the drug application. Each dot indicates a single neuron pair.</p

Putative pyramidal neurons are more affected by SKF38393 than putative interneurons.

<p><b>A.</b> The mean SW-locked probability of cells with the top 20% SW-locked probability (left) and that of the bottom 80% SW-locked probability (right). <b>B.</b> The mean amplitude of calcium transients of the top 20% SW-locking cells (left) and that of the bottom 80% cells (right; *<i>P</i> = 0.016, <i>t</i>₇ = 3.14, paired <i>t</i>-test). <b>C.</b> A Venn diagram of the population of cells that participated in at least one SW event during the 3-min periods before and after the application 30 µM SKF38393 for the top 20% SW-locking cells (left) and the bottom 80% cells (right).</p

SKF38393 preserves SWs-participating neurons.

<p><b>A.</b> A Venn diagram of the population of cells that participated in at least one SW event during the 3-min periods before and after the application of control aCSF (left) or 30 µM SKF38393 (right). The values indicate the percentage of cells involved in the corresponding states to the total cells. The two populations overlapped significantly. Control: <i>P</i> = 1.85×10⁻⁵<i>versus</i> the chance level (21.2%), <i>Z</i> = 4.28; SKF38393: <i>P</i> = 1.62×10⁻⁸ versus the chance level (16.9%), <i>Z</i> = 5.65, Z-test for a proportion. <b>B.</b> Relationship of the frequencies of SW-locked activities of individual neurons before and after control aCSF (left) or 30 µM SKF38393 (right) administration. Each dot indicates a single neuron. Control: <i>R</i>² = 0.57, <i>P</i> = 5.0×10⁻⁴⁸, <i>t</i>₂₅₁ = 18.3; SKF38393: <i>R</i>² = 0.64, <i>P</i> = 3.7×10⁻⁸³, <i>t</i>₃₆₄ = 25.5, <i>t</i>-test of a correlation coefficient. <b>C.</b> The mean percentage of cells that participated in a single SW event to the total cells before and after aCSF or SKF38393 administration. Each gray dataset represents a single slice. Control: <i>P</i> = 0.93, <i>t</i>₄ = 0.098; SKF38393: <i>P</i> = 0.25, <i>t</i>₇ = 1.23, paired <i>t</i>-test. Data are the means ± SEMs of 5 or 8 slices. <b>D.</b> Comparison of the mean amplitudes of SW-locked calcium transients from 150 and 199 SW participants before and after the drug application. Control: <i>R</i>² = 0.091, SKF38393, <i>R</i>² = 0.00025, <i>P</i> = 0.13, <i>Z</i> = 1.53, <i>Z</i>-test for two correlation coefficients. Each dot indicates a single neuron.</p

SKF38393 increases the repertoires of SW-relevant firing patterns.

<p><b>A.</b> Representative spatiotemporal patterns of SW-locked activities in 136 SW events during the observation period of 3 min. <b>B.</b> The correlation coefficients between patterns of SW-participating neurons were calculated for all possible SW pairs. The representative correlation matrix was obtained from 136 SWs shown in A. <b>C.</b> Cumulative distribution of the correlation coefficients before (green) and after (orange) perfusion with control aCSF [left, 795 SW events (before) and 1,076 (after) from 8 slices] and SKF38393 [right, 795 SW events (before) and 1,076 (after) from 8 slices]. <b>D.</b> The affinity propagation algorithm separated 136 SW events in B. into 26 SW subgroups, indicated by different colors. <b>E.</b> The mean number of the SW subgroups before and after the application of control aCSF (left) and SKF38393 (right). Each gray dataset indicates a single slice. *<i>P</i> = 0.030, <i>t</i>₇ = 2.25, paired <i>t</i>-test. Data are the means ± SEMs of 5 or 8 slices. <b>F.</b> The same analysis as E was repeated a time window between −50 and +50 ms relative to the SW peak (left). The mean numbers of the SW subgroups before and after the application of control aCSF (left) and SKF38393 (right) are shown in the bar graph. *<i>P</i> = 0.036, <i>t</i>₇ = 2.12, paired <i>t</i>-test. Data are the means ± SEMs of 8 slices. <b>G.</b> The relationship between the number of SW-participating neurons and of SW patterns. Data obtained from the same slice are connected with black line. Before (green); <i>R</i>² = 0.21, <i>P</i> = 0.24. After (orange); <i>R</i>² = 8.3×10⁻⁴, <i>P</i> = 0.95.</p