Study of the DiI method accuracy on the example of the afferent connections of the Habenula in P6 F1-C57Bl6 / CBA mice

Abstract

Klepukov A), embryology@altravita.ru, ABSTACT DiI (1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate) is a lipophilic carbocyanine stain diffusing in the plasma membrane and coloring the cell as a whole. The DiI injection method is an alternative to stereotaxis in the study of the neural connections of higher vertebrates. However, despite a number of studies performed to identify the neural connections of embryos and fetuses of different animals, the question of the accuracy of the DiI method remains open. The main goal of this study is to investigate the accuracy of the DiI method by examining the afferent Habenula (Hb) connections in P6 F1-C57Bl6/CBA mice. Hb is a paired structure of the intermediate brain, consisting of two nuclei -the medial (MHb) and lateral (LHb). At P6, the main Hb tracts are already formed but not yet myelinated. To estimate the accuracy of the method, two groups of cases with different types of application on different Hb nuclei were compared: bilateral applications of several DiI crystals -group 1 (26 applications); Unilateral applications of a single crystal DiI -control group 2 (7 applications). It is known that MHb is strictly innervated by Triangular septal nuclei (TS) and Septofimbrial septal nuclei (SFi); LHb is strictly innervated by the Lateral preopteic area (LPA). Any deviation from this scheme is a false positive result. When applying the marker to different Hb nuclei in group 1, the number of successful cases without deviations was -0%; in group 2 (control) -28.57%. Unilateral applications of a single DiI crystal are more accurate. However, there are still a number of unavoidable problems, such as the fragmentation or loss of a single DiI crystal during application and the super-strong diffusion of the marker at the application site. Given this, the overall accuracy of the DiI injection method, even in the form of a single crystal, remains low.Keywords: crystals/crystal DiI, afferent connections of the Habenula; bilateral applications; unilateral applications; P6 mice F1 -C57Bl6 / CBA.