Microscopy and data acquisition

Microscopy was performed using an Axiovert 200 M microscope equipped with an ApoTome device for near confocal imaging, AxioCam MRm digital camera and AxioVision software v4.8.2 (all from Carl Zeiss Microscopy). The latter includes the MosaiX module that permits the sample area to be scanned in order to generate one single large image.

4.8.1 Analyzing interneurons on coronal sections

MosaiX-images of coronal sections were acquired using the ApoTome technology with a 10x Plan-Apochromat objective with a numerical aperture of 0.45. All samples were matched for exposure times. In adult mice, the subdivision of the medial prefrontal cortex into Cg1, PrL and IL, as well as into upper and deep cortical layers (corresponding to layers 1 to 3 and 5 + 6) was achieved according to Paxinos and Franklin (2001). Cells were counted by visual inspection with the aid of the interactive event counting tool of AxioVision software. The estimated cell numbers were related to the area of the respective region. Cell densities were estimated per section and respective cell densities per animal were calculated by estimating the mean of up to six sections. Extents of migratory streams on embryonic coronal sections were analyzed by estimating the distance of the very last interneuron in a migratory stream to the pallial / subpallial boundary.

4.8.2 Densitometric quantification of polySia on sections

PolySia levels in the area of migrating interneurons on coronal sections were quantified by analyzing images acquired using the ApoTome technology with a 10x Plan-Apochromat objective. Exposure times were identical among all samples. Ten optical sections with a distance of 0.55 µm along the z axis and each with a thickness of 5.1 µm were acquired and merged into one image using AxioVision software. These images were exported as TIFF files in 16 bit grayscale to imageJ software v1.44p (Schneider et al., 2012). Background was subtracted using the rolling ball method, the radius set to 10 pixels. The area of the intermediate zone migratory stream was outlined and analyzed, yielding mean grey values.

Strongly polySia-positive blood vessels were excluded from analysis.

PolySia levels on somata of calbindin-positive cells were quantified as described above.

Images were acquired using a 63x Plan-Apochromat oil-immersion objective with a numerical aperture of 1.4. Optical sections had a distance of 0.25 µm and a thickness of 0.81 µm.

4.8.3 Counting of GFP-positive cells in slice- and co-cultures

MosaiX-images of slice cultures were acquired by ApoTome technology using a 5x Plan-Neofluar objective with a numerical aperture of 0.15. All samples were matched for exposure times. With the aid of AxioVision software, curves with ascending distances of 100 µm starting from the intersection of strongly GFP-positive ganglionic eminence and pallium were drawn and overlaid into the image. The curves hereby resembled the shape of the intersection. This way, bins of ascending distances to the subpallial / pallial boundary were created and numbers of GFP-positive cells within these bins were estimated.

MosaiX-images of slice cultures were acquired by ApoTome technology using a 10x Plan-Apochromat objective. Numbers of GFP-positive cells were estimated as described before.

The intersection of GFP-positive MGE and GFP-negative host tissue served as starting point for the creation of bins.

4.8.4 Estimation of migration in MGE explant cultures

Mosaix-images of MGE-explant cultures were acquired using a 5x Plan-Neofluar objective.

At the beginning of the experiment (t=0), micrographs were acquired by phase contract microscopy. After 3 days in vitro, Mosaix-images of GFP-fluorescence were acquired with using ApoTome technology. With the aid of AxioVision software, the area of the explant was estimated at t=0. Images of cultures after 3 days in vitro were exported in TIFF format in 8 bit grayscale and the area covered by GFP-positive cells was analyzed using imageJ software.

The required threshold-settings were adjusted identically in all analyzed samples and scaling of the images was set according to the original images from AxioVision software. Areas at t=0 were subtracted from the respective areas at 3 days in vitro, resulting in areas covered by migrating cells during cultivation of the explant.

4.8.5 Analysis of interneuron leading processes

Images of MGE primary cultures were acquired using the ApoTome technology with a 20x Plan-Apochromat objective with a numerical aperture of 0.8. With the aid of AxioVision software, extents of leading processes of GFP-positive cells were estimated. Lengths were determined by tracing the process from the tip of the growth cone to its onset defined as the site of somal constriction. Only cells with a clearly discernible leading process were analyzed.

4.8.6 Live imaging

Live imaging was performed at 37°C and 5%CO2 using a humidified incubator (Pecon, Erbach, Germany) mounted to the microscope. Using multiwell imaging slides, up to 6 independently cultured slices were analyzed in one experiment. Time lapse sequences were generated by acquiring images with a 10x Plan-Apochromat objective at an interval of 2 min for up to 14 hours. For the quantification of interneuron precursor velocity, a sequence of 180 min starting 4 hours after the onset of image acquisition was analyzed. Only cells visible throughout the entire sequence were used for analysis and migratory paths were reconstructed using the AxioVision software. The resulting distances were used to estimate the velocity of migrating cells. Binning of velocities was achieved by using the frequency distribution function of GraphPad Prism software v4 (GraphPad Software Inc., La Jolla, CA, USA). In order to summarize the direction of migration for one experimental condition, all obtained paths were centered to a common starting point. The end points of the migratory paths were marked in AxioVision software and TIFF images were exported to imageJ in two color mode. Within imageJ software the coordinates of the marks were estimated and exported to GraphPad Prism software to create a graph illustrating the final position of every cell in relation to the starting point.

4.8.7 Documentation of in situ hybridization

MosaiX-images were acquired using brightfield-microscopy with a 10x Plan-Apochromat objective. Merged images of micrographs were created using the transparency and overlay functions in CorelDraw software X3 (Corel, Fremont, CA, USA).