Method for Enhancing Cell Penetration of Gd³⁺-based MRI Contrast Agents by Conjugation with Hydrophobic Fluorescent Dyes

Gadolinium ion (Gd³⁺) complexes are commonly used as magnetic resonance imaging (MRI) contrast agents to enhance signals in <i>T</i>₁-weighted MR images. Recently, several methods to achieve cell-permeation of Gd³⁺ complexes have been reported, but more general and efficient methodology is needed. In this report, we describe a novel method to achieve cell permeation of Gd³⁺ complexes by using hydrophobic fluorescent dyes as a cell-permeability-enhancing unit. We synthesized Gd³⁺ complexes conjugated with boron dipyrromethene (<b>BDP-Gd</b>) and Cy7 dye (<b>Cy7-Gd</b>), and showed that these conjugates can be introduced efficiently into cells. To examine the relationship between cell permeability and dye structure, we further synthesized a series of <b>Cy7-Gd</b> derivatives. On the basis of MR imaging, flow cytometry, and ICP-MS analysis of cells loaded with <b>Cy7-Gd</b> derivatives, highly hydrophobic and nonanionic dyes were effective for enhancing cell permeation of Gd³⁺ complexes. Furthermore, the behavior of these <b>Cy7-Gd</b> derivatives was examined in mice. Thus, conjugation of hydrophobic fluorescent dyes appears to be an effective approach to improve the cell permeability of Gd³⁺ complexes, and should be applicable for further development of Gd³⁺-based MRI contrast agents

Cofilin1 Controls Transcolumnar Plasticity in Dendritic Spines in Adult Barrel Cortex

<div><p>During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure <i>in vitro</i>, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.</p></div

Current amplitude dependence of the response magnitude of the cerebellar activation.

<p>(<b>a</b>) Average time course of the cerebellar activation in response to an individual EM block. The shaded region indicates the EM block. For each time course, the baseline signal [mean of 2 frames (5 sec) before the onset of EM block] was subtracted before averaging. Error bars indicate standard error (SE). Red, 750 µA (240 blocks). Green, 500 µA (264 blocks). Blue, 250 µA (240 blocks). (<b>b</b>) Response magnitude for each current amplitude. Colored lines indicate data for individual monkeys (gray solid line, Monkey 1; gray dotted line, Monkey 2). Error bars indicate SE. *, <i>P</i><0.02. **, <i>P</i><0.0002. ***, <i>P</i><10⁻⁸.</p

List of BOLD activations in the cerebellum.

<p>List of the coordinates of the peaks of EM-evoked BOLD activations in the cerebellum for two monkeys (Monkey 1, 250 µA, 30 runs; Monkey 2, 500 µA, 9 runs). Significance level was set at <i>P</i><0.05 (corrected). Activated regions with volumes ≥6 mm³ (2.1 original voxels) are included. Cb4, cerebellar lobule 4. Cb5, cerebellar lobule 5. Cb6, cerebellar lobule 6. Cop, copula pyramidis. Crus2, crus2 of ansiform lobule. DPFl, dorsal paraflocculus. PM, paramedian lobule. Anatomical areas are labeled by referring to the Paxinos et al. brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047515#pone.0047515-Paxinos1" target="_blank">[22]</a>.</p

BOLD activations in the cerebellum induced by electrical stimulation of S1.

<p>(<b>a</b>)–(<b>b</b>) A representative <i>t</i>-score map of BOLD activation in Monkey 1 in one session (250 µA, 30 runs). (a) BOLD activation at the site of EM. In Monkey 1, right S1 was stimulated (arrow). (b) BOLD activations in the cerebellum. Cb5, cerebellar lobule V. Cop, copula myramidis. (<b>c</b>)–(<b>d</b>) A representative <i>t</i>-score map of BOLD activation in Monkey 2 in one session (500 µA, 9 runs). Conventions are the same as in (a) and (b). (c) BOLD activation at the site of EM. In Monkey 2, left S1 was stimulated (arrow). (d) BOLD activations in the cerebellum.</p

Efficiency and specificity of CFL1 KD through miR-CFL1.

<p>(A) CFL1 mRNA KD efficiency of two miRNAs (miR-CFL1_1 and miR-CFL1_2) targeted against different sequences within the CFL1 gene, assessed by using CFL1-overexpressing HEK 293T-cells. CFL1 mRNA levels were normalized to those of the miR-Neg group. <i>n</i> = 3 for all groups; miR-CFL1_1, <i>p</i> = 2.7 × 10^-8; miR-CFL1_2, <i>p</i> = 2.8 × 10^-8 versus miR-Neg, Dunnett’s multiple comparison test. (B) KD efficiency of miR-CFL1_1 and miR-CFL1_2 for endogenous CFL1 protein, assessed by using PC-12 cells. “WT” (wild type) indicates PC-12 cells that were not infected with lentivirus. (C) Two neighboring coronal sections obtained from a miR-CFL1_1-expressing rat are shown, one stained with an antibody against CFL1 (left) and the other stained with an antibody against NeuN (right). The eYFP fluorescence image (middle) was obtained from the NeuN-stained section. Scale bar, 300 μm. (D) Magnified view of the rectangular region indicated in (C). Scale bar, 150 μm. (E, F) Same as (C and D), but of two neighboring coronal sections derived from a miR-Neg virus-injected rat. (G, H) Confocal images of eYFP+ or eYFP− region in a coronal section obtained from a miR-CFL1_1-expressing rat stained with antibodies against NeuN (blue) and CFL1 (red). Scale bar, 50 μm. (I) Percentage of CFL1+ cells in NeuN+ cells measured in miR-CFL1_1- or miR-Neg-expressing rats. <i>n</i> = 3 for all groups. *<i>p</i> = 0.0003, t-test with Bonferroni’s correction. (J) Effects of miR-CFL1 on mRNA expression of genes related to CFL1, assessed in PC-12 cells. <i>n</i> = 3 for all groups. miR-CFL1_1 of CFL1, <i>p</i> = 2.6 × 10^-7; miR-CFL1_2 of CFL1, <i>p</i> = 5.2 × 10^-7 versus miR-Neg, Dunnett’s multiple comparison test. (K) Effects of miR-CFL1 on expression of ADF protein in PC-12 cells. (L) Three successive coronal sections obtained from a miR-CFL1_1-expressing rat are shown, one stained with an antibody against ADF (middle) and another stained with an antibody against CFL1 (right). Scale bar, 300 μm.</p

Effects of CFL1 expression rescue on impaired experience-dependent plasticity.

<p>(A) Design of three mutant CFL1s that are resistant to miR-CFL1_1 (resCFL1s). Seven or eight nucleic acids within the target sequence of miR-CFL1_1 were mutated such that amino acid sequence did not change. (B) An <i>in vitro</i> test of CFL1 expression rescue by each resCFL1 in rat CFL1 (WT)- and miR-CFL1_1-expressing HEK293T cells. The mock group expressed only WT CFL1. <i>n</i> = 4 for all groups. resCFL1_1, <i>p</i> = 5.8 × 10^-6; resCFL1_2, <i>p</i> = 2.2 × 10^-5; resCFL1_3, <i>p</i> = 1.3 × 10^-4 versus miR-CFL1_1 group, Tukey-Kramer’s multiple comparison test. (C) An <i>in vitro</i> test of the effect of miR-CFL1_1 on resCFL1_1 expression. <i>n</i> = 3 for both groups. <i>p</i> = 0.30, <i>t</i>-test. (D) A schematic diagram of the bicistronic lentiviral vector that co-expresses resCFL1_1 and mCherry via a P2A peptide. (E) Targeted injection of Lenti-CaMKIIα-ChR2-eYFP-miR-CFL1_1 and Lenti-CaMKIIα-mCherry-P2A-resCFL1 to D2 column identified with intrinsic signal imaging induced focal expression of ChR2-eYFP and mCherry. Scale bar, 500 μm. (F) Fluorescent images of a coronal section infected with the two vectors. Scale bar, 300 μm. (G) Confocal images of an infected area showing co-expression of ChR2-eYFP and mCherry in L2/3 neurons. Scale bar, 20 μm. (H) A representative raster plot (100 trials are shown in horizontal row) and peristimulus time histogram (PSTH) of a putative ChR2+ neuron recorded from L2/3 in D2 column of the rat showed in E. (I) A representative raster plot (50 trials) and PSTH of the same neuron with (H), showing responses to D1 whisker deflections. (J) Comparison of average responses recorded from ChR2+ neurons in D2 L2/3 of deprived rats expressing miR-CFL1_1 and resCFL1 with those recorded from ChR2+ neurons in deprived rats expressing only miR-CFL1_1 and those recorded from WT deprived rats. Data of miR-CFL1_1 deprived and WT deprived groups were the same with those shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002070#pbio.1002070.g003" target="_blank">Fig. 3D</a>. <i>n</i> = 17 units (from three rats) for the miR-CFL1_1+resCFL1 deprived group. *<i>p</i> = 0.0069, Tukey-Kramer’s multiple comparison test.</p

Schematic illustration of the circuit-specific cofilin action on barrel map plasticity.

<p>Cross-sectional view of the D1 and D2 barrel columns. L2/3 neurons in the D2 column exhibit a CFL1-dependent neuronal response increase to horizontal transcolumnar inputs from the spared D1 column. By contrast, the response decrease to ascending intracolumnar inputs from L4 is CFL1 independent. In the distal portion of the supragranular layer of the D2 column, spine densities of dendrites receiving transcolumnar inputs from the D1 column increase in a CFL1-dependent manner during sensory deprivation.</p

Effects of CFL1 KD on spine density during sensory deprivation.

<p>(A, B) Lentiviral vectors employed for co-expressing eGFP and miRNAs (A) and for expressing tdTomato (B). (C) The D1 and D2 barrel columns were identified via intrinsic signal optical imaging. (D) Virus injection was targeted to L2/3 of the D1 and D2 columns. (E) A representative parasagittal section showing expression of tdTomato (D1) and eGFP (D2). Scale bar, 300 μm. (F) Magnified view of an eGFP-expressing region in the parasagittal section shown in (E). Scale bar, 100 μm. (G) Confocal images of a rectangle region shown in (F). Scale bar, 20 μm. (H) A representative dendritic branch in the D2 column is shown that made a putative synaptic connection with a tdTomato+ D1 axonal bouton. Dendritic spines were counted that were localized at a distance less than 15 μm from an identified putative synaptic connection. A magnified view of the putative synaptic connection is shown in the inset. Scale bar, 10 μm. (I) Representative images of the dendritic branches within the distal portion in the D2 column. Scale bar, 10 μm. (J) Spine densities measured in the distal portion. <i>n</i> = 18, 17, 19, and 19 branch segments for miR-Neg non-deprived (ND), miR-Neg deprived (D), miR-CFL1_1 ND, and miR-CFL1_1 D groups, respectively. WT ND, <i>p</i> = 1.1 × 10^-4; miR-CFL1_1 ND, <i>p</i> = 1.8 × 10^-5; miR-CFL1_1 D, <i>p</i> = 0.0027 versus WT D group, Tukey-Kramer’s multiple comparison test. (K) Cumulative frequency histogram of spine density. WT ND, <i>p</i> = 0.037; miR-CFL1_1 ND, <i>p</i> = 3.6 × 10^-4; miR-CFL1_1 D, <i>p</i> = 3.6 × 10^-4 versus WT D group, Kolmogorov-Smirnov test with Bonferroni’s correction. (L−N) Same as (I−K) but of dendritic branches measured within the proximal portion. <i>n</i> = 14, 18, 16, and 10 branch segments for miR-Neg ND, miR-Neg D, miR-CFL1_1 ND, and miR-CFL1_1 D groups, respectively. WT ND, <i>p</i> = 0.86; miR-CFL1_1 ND, <i>p</i> = 0.97; miR-CFL1_1 D, <i>p</i> = 0.93 versus WT D group, Tukey-Kramer’s multiple comparison test.</p

Simultaneous fMRI and electrical stimulation of S1.

<p>(<b>a</b>) Left panel, schematic drawing of a monkey brain with a microelectrode inserted in S1. Right panel, anatomical MRI image (FLASH) showing a monkey brain with a microelectrode inserted. ele, microelectrode. cs, central sulcus. ips, intraparietal sulcus. (<b>b</b>) Coronal sections of a representative <i>t</i>-score map of BOLD activation in Monkey 1 in one session (250 µA, 30 runs). In Monkey 1, right S1 was stimulated. A5, the area 5. Cb, cerebellum. S1, primary somatosensory cortex. S2, secondary somatosensory cortex. Thal, thalamus. stim, the site of EM. (<b>c</b>) Time courses of BOLD activations in S1 and Thal (30 runs, 240 EM blocks). Baseline signal [mean of 2 frames (6 sec) before the onset of EM block] was subtracted before averaging. White bars indicate 30 sec blocks of EM.</p