Growth cone morphology, adhesiveness and motility

An essential evaluation was to prove how the cortical neurons that lack M6 proteins do behave while being in culture, as the prior results demonstrated an altered behaviour of the neurons in their neurite outgrowth and in their growth cone function. Therefore, primary cortical neurons of E17 wild-type and Gpm6a^null*Gpm6b^null mice were subjected to in vivo

reflection microscopy was used. As M6 proteins seem to be especially important in the growth cone, the analysis focused on this structure. The individual growth cones were imaged during ten minutes and an image was acquired every three seconds, so that for every growth cones were 201 frames which were converted into a video (Fig. 23A). From the wild-type cortical neurons nine growth cones were imaged, and ten in the Gpm6a^null*Gpm6b^null ones. The imaging acquisition was chosen to be of a period of ten minutes, as it had already been assessed that the Gpm6a^null*Gpm6b^null cortical neurons have an impaired neurite extension, so that there would be no masking of the effect of the reduced neurite extension in their motility properties.

Figure 23. Morphometry of in vivo imaged cortical neuron growth cones.

A) Cortical neurons of E17 WT and Gpm6a^null*Gpm6b^null (dKO) mice were cultured for 2 DIV. In vivo imaging of their growth cones during 10 min, performing one image every 3 seconds. Depicted is the first image for one growth cone of each genotype. Scale bars = 5 µm.

B) Representation of the normalized mean ratio of the perimeter against the total area. No significant differences could be observed.

C) Representation of the normalized mean total area. No significant differences could be observed.

The first approach was to analyse the morphometrical properties. The perimeter of each growth cone in each frame was determined, as well as the total surface. When comparing the normalized values for the ratio of the perimeter against the total area (Fig. 23B), as well

as the total area per se (Fig. 23C), no difference could be assessed. This was proven by realizing an ANOVA with repeated measurements (Greenhouse-Geisser) (perimeter / total area: F = 1.287, P = 0.287; total area: F = 0.411, P = 0.758). Thus, growth cones of cortical neurons that lack M6 proteins do have the same morphometrical properties than wild-type ones.

Because of employing reflection microscopy, the various levels of attachment to the glass slide could be differentiated. Thereby, the following analysis was based on the different adhesion levels. The area of the growth cones was divided into “adhesive” and “non adhesive” according to the intensity levels on each single image (Fig. 24A). When performing the ratio of the normalized data of the “adhesion area” Vs the “non adhesion area” no differences could be proven (Fig. 24B, C). This was confirmed by means of an ANOVA with repeated measurements (Greenhouse-Geisser) (adhesion area / total area: F = 1.150, P = 0.340). Consequently, the adhesive properties are also not altered when analysing neuronal growth cones that lack chronically neuronal M6 proteins.

Figure 24. Adhesiveness of in vivo imaged cortical neuron growth cones (cont.)

Figure 24. Adhesiveness of in vivo imaged cortical neuron growth cones (cont.).

A) Cortical neurons of E17 wild-type and Gpm6a^null*Gpm6b^null (dKO) mice were cultured for 2 DIV. The growth cones were imaged in vivo during 10 min, achieving one image every 3 seconds. According to the intensity levels, the growth cone area was classified into “adhesion” (red) and “non adhesion” (green) areas. Depicted are four example images of each genotype, chosen every second frame from the first one on. The time passed is indicated on each image. Scale bars = 5 µm.

B) Representation of the normalized ratio of the adhesive area against the total area. No significant differences could be observed.

C) Representation of the normalized mean adhesive area against the total area. No significant differences could be observed.

Furthermore, the examination of the videos was performed in such a manner that the motility of the growth cones could be investigated, as this is an essential factor of the growth cone functionality. Between each frame of each single video, the differences form one to the next frame were analysed in such a way that the extension and retraction areas could be assessed (See Fig. 7). As the areas occupied by the growth cone in one frame were subtracted form the prior one, extension was evaluated as values minor than zero and retraction as values larger than zero. If there would be no difference, the sum would be zero.

When comparing the growth cones regarding the extension (Fig. 25A-B) and retraction (Fig.

25C-D) levels no differences could be observed between wild-type and Gpm6a^null*Gpm6b^null. This is obvious when observing the normalized mean alteration of the sum of the retraction and extension values (Fig. 25E), as it is basically identical for the growth cones of wild-type and Gpm6a^null*Gpm6b^null cortical neurons. Additionally, when plotting the normalized differences of retraction and extension (Fig. 25F) no differences could be observed. An ANOVA with repeated measurements (Greenhouse-Geisser) was performed and validated this (retraction + extension: = 0.992, P = 0.494; retraction-extension: F = 1.184, P = 0.286).

Yet again, there is no difference of the in vivo behaviour of the Gpm6a^null*Gpm6b^null cortical neuron growth cones. The way they retract and extend over a period of 10 minutes is equivalent.

Figure 25. Motility of in vivo imaged cortical neuron growth cones.

Cortical neurons of E17 WT and Gpm6a^null*Gpm6b^null (dKO) mice were cultured for 2 DIV. Growth cones were image in vivo during 10 min, performing one image every 3 seconds. The differences in area occupied by the growth cone were assessed between each frame of each single video, by subtracting the occupied area of one frame with the prior one. Thereby positive values measure retraction and negative values measure extension. If there is no net movement, the value would be zero.

A-B) Representation of the normalized extension values for WT (A) and dKO (B) growth cones.

C-D) Representation of the normalized retraction values for WT (A) and dKO (B) growth cones.

E) Representation of the normalized mean sum of the retraction and extension values in WT (black) and dKO (red) growth cones.

F) Representation of the normalized levels of retraction and extension values in WT (black) and dKO (red) growth cones.

This in vivo analysis demonstrates that the short-term morphometry, adhesion and motility is not altered in the growth cones of Gpm6a^null*Gpm6b^null cortical neurons.