Exposure occasions were kept constant to allow comparison between individual slices. the migration of GCs in a distance-dependent manner. In addition, quantitative Western blot analysis revealed that KA treatment affects the Reelin transmission transduction pathway by increasing intracellular adaptor protein Disabled-1 synthesis and reducing the phosphorylation of cofilin, a downstream target of the Reelin pathway. Both events were normalized by addition of recombinant Reelin fragments. Finally, following neutralization of Reelin in healthy OHSC by incubation Rabbit Polyclonal to GCVK_HHV6Z with the function-blocking CR-50 Reelin antibody, GCs started Bromfenac sodium to migrate without any direction preference. Together, our findings demonstrate that normotopic position of Reelin is essential for the maintenance of GC lamination in the dentate gyrus and that GCD is the result of a local Reelin deficiency. mouse (DArcangelo et al., 1995; Hirotsune et al., 1995) shows a disorganized granule cell layer (GCL), reminiscent of GCD (Frotscher et al., 2003). Moreover, GCD formation has been shown to be accompanied by a loss of Reelin-producing neurons in the hippocampus of MTLE patients (Haas et al., 2002; Liu et al., 2020) and in rodent epilepsy models (Heinrich et al., 2006; Gong et al., 2007; Antonucci et al., 2008; Duveau et al., 2011; Orcinha et al., 2016). A local rescue of GC lamination has been achieved by infusion of recombinant Reelin into the mouse hippocampus during epileptogenesis (Mller et al., 2009) and by addition of recombinant Reelin in kainate (KA)-treated organotypic hippocampal slice cultures (OHSC; Orcinha et al., 2016). Conversely, neutralization of Reelin in the healthy mouse hippocampus by infusion of the function-blocking CR-50 antibody caused a local widening of the GCL (Heinrich et al., 2006). GCD can be induced in OHSCs by KA application (Tinnes et al., 2011, 2013; Chai et al., 2014). In this model, differentiated GCs have been shown to migrate via somal translocation (Murphy and Danzer, 2011; Chai et al., 2014), but so far the precise mechanism has remained unclear. In the present study, we used KA-treated OHSC from transgenic mice expressing enhanced green fluorescent protein (eGFP) in differentiated GCs (1) to monitor the movement behavior of individual GCs in detail by live cell video microscopy, (2) to investigate which part of the Reelin molecule is usually efficient in GCD rescue, (3) whether the site of the Reelin loss matters, and (4) whether Reelin is needed for the maintenance of lamination in the healthy hippocampus. We present evidence that differentiated GCs actively migrate toward the Reelin-free hilar region and this motility can be prevented by application of the recombinant N-terminal or the central Reelin fragment. In addition, we show that placement of Reelin-coated beads into the hilus significantly stops the aberrant migration of GCs after KA exposure, and that neutralization of Reelin by the CR-50 antibody causes locomotion of GCs in healthy OHSC. Materials and Methods Animals Experiments were performed with transgenic Thy1-eGFP mice, in which eGFP is usually expressed under the control of the Thy1 promoter in a subpopulation of differentiated GCs in the dentate gyrus (RRID: IMSR_JAX:007788, M-line, C57BL/6 background, Feng et al., 2000). Bromfenac sodium Mice were bred at the Center for Experimental Models and Transgenic Support (CEMT), University or college of Freiburg, and kept in a 12 h light/dark cycle at room heat (RT) with food and water (DIV) to allow full eGFP expression (Chai et al., 2014) which was checked using an inverted epifluorescence microscope (Olympus CKX41 with U-RFL-T, Olympus). Incubation medium was changed every second day. Live Cell Imaging Organotypic hippocampal slice cultures were prepared as explained above and cultivated for 7 C 18 DIV for the different live cell imaging experiments. Immediately before imaging, OHSC were dealt with as follows: (1) to monitor the migration behavior of individual GCs, OHSC were treated with KA (10 M; Tocris) for 45 min, followed by addition of new medium with or without recombinant Reelin fragments (1 nM); Reelin fragments were produced as previously explained Bromfenac sodium in Orcinha et al. (2016); (2) to study the influence of Reelin in normotopic position within the dentate gyrus, OHSC were treated with KA (10 M) for 45 min.