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Ethics Statement: The handling of the mice and the experimental procedures were conducted in accordance with European, national and institutional guidelines for animal care.

1. Electroporation

Note: electroporation was performed as previously published. Therefore, the method is only described briefly in the following ( Figure 1 ).

2. Transfection of Specific Cerebral Areas

Note: The position of the positive pole is shown as the angle between the vertical centerline through the ventricle filled with the DNA solution (the right or third ventricle) and the position of the positive pole. When filling the left ventricle the positions are mirror-inverted. In Figure 4 the positions of the ear primordial are shown, which provide important reference points.

3. Perfusion Fixation

4. Immunohistochemistry

Figure 2A Figure 3 Figure 2B Table 1 Figure 2C Figure 2D

Figure 2 , shows examples for the specific electroporation of the regions developing into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. The results of the transfections are shown next to the recommended angle ( Figure 2 ). For better visualization of the angles the position of the electrode (0.5 mm) are also shown on an embryo ( Figure 5 , embryo , embryo in amniotic sac, dissected embryo). In order to provide an overview Table 1 summarized the relevant facts for specific transfection.

Further, it was demonstrated that the size of the electrode plays a crucial role for the specificity ( Figure 6 ). Larger size of the electrode paddles leads to larger transfected areas, as exemplary shown in Figure 6 . Further, electrodes that are too large cover too much of the embryo, thereby enhancing the risk of affecting the embryos heart rhythm ( Figure 7 ). For example a 10 mm electrode paddle covers the whole E12 embryo ( Figure 7 ).

Figure 1. electroporation. Basic steps of the performance of the electroporation are shown. ( A ) Shows the whole setup of the surgery, with the mouse placed on a hotplate already anesthetized with isoflurane. ( B ) The disinfection of the surgical area is a crucial step before opening the abdominal cavity ( C ) of the mouse. ( D ) The DNA-containing solution is colored with Fast Green, which enables the visualization of successful injection ( E , F ). Successful injection into a lateral ventricle results into crescent-shaped spot. ( G ) After the injection the appropriate voltage is applied via forceps type platinum electrodes. ( H ) After carefully replacing the uterus into the abdominal cavity the surgical wound is sutured. Clearance Deals Prada Embellished Ballet Flats Free Shipping Websites x29Pj

Figure 2. Angle-map for electroporation of specific areas in the murine cortex, hippocampus, lateral septal nucleus, striatum, thalamus and hypothalamus. The appropriate positions for the positive and negative pole, are indicated in the schematic drawing. For ( A-C ) the positions are indicated for injecting the DNA-solution into the right ventricle. For injections into the left ventricle, the angles are mirror-inverted. The specific transfections were histologically verified. ( A ) Shows the positions of the positive and negative pole for transfecting cortical areas (the retrosplenial cortex (RSC), the motor cortex (MC), the somatosensory cortex (SSC) and the piriform cortex (PC). ( B ) Shows the positions of the positive and negative pole for transfecting the hippocampal formations (the cornu ammonis 1-3 (CA1, CA2, CA3) and the dentate gyrus (DG). ( C ) Shows the positions of the positive and negative pole for transfecting the striatum (STR) and the lateral septal nucleus (LS). ( D ) Shows the appropriate positions of the electrodes for transfecting the thalamus (TH) and the hypothalamus (HY). Adapted from Baumgart J. Grebe N., 2015 . NUMIS Platform heels black How Much Uf99Ur7gC

Figure 3. Puncture site. The optimal position for the injection of the DNA solution isapproximately 0.75 mm coronal from the lambdoidal suture and 0.5 mm lateral from the sagittal suture. Robert Clergerie Clergerie Paris Raffia Oxford Wedges Professional For Sale Official Online Buy Cheap 2018 Unisex Buy Cheap Pre Order OVpH6kpWq5

Figure 4. Position of the ear primordium. The position of the ear primordium is clearly defined (arrow). 212 hitop sneakers Cheap Pay With Visa Amazing Price Sale Online 6SiLVrihUy

Figure 5. Visualization of the angles on an embryo. For better visualization the appropriate positions for the positive (indicated with a blue plus sign) and the negative pole for specific transfections of the cortical areas (retrosplenial cortex, motor cortex, somatosensory cortex, piriform cortex), the hippocampal areas (cornu ammonis (CA) 1-3, dentate gyrus (DG)), the lateral septal nucleus, the striatum, the thalamus and hypothalamus are shown on an embryo , an embryo in the amniotic sac and on a dissected embryo (all E15,5). Cheap Price From China Salvatore Ferragamo Vara PeepToe Wedges Huge Surprise Sale Online Clearance Genuine Cheap Sale Genuine Fake O8Qsj77

Figure 6. Specificity of transfection depending on the electrode size. Increased size of the electrode paddle reduces the specificity of the transfection. This is especially obvious in younger embryonic stages (E12, top), but also visible in late embryonic stages (E17, bottom). Adapted from Baumgart J. Grebe N., 2015 . Please click here to view a larger version of this figure.

Figure 7. Size of the electrode paddle compared to the size of an E12 embryo. Electrodes that are too large cover too much of the embryo and therefore increase the risk of affection of the heart rhythm. For example, a 10 mm electrode (on the right) covers the whole E12 embryo and therefore is not recommended, even if specificity is not the primary goal. Adapted from Baumgart J. Grebe N., 2015 . Please click here to view a larger version of this figure.

Table 1: Summary of results. Included in this table is all relevant information for specific transfection of the described cortical areas. It contains details about the targeted region, the angle, the position of the electrode, the embryonic stage, the applied amount and concentration of the DNA solution, the voltage and the size of the electrode.

In utero

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