Phosphodiesterase 2 Inhibitors Promote Axonal Outgrowth in Organotypic Slice Co-Cultures

Abstract

The development of appropriate models assessing the potential of substances for regeneration of neuronal circuits is of great importance. Here, we present procedures to analyze effects of substances on fiber outgrowth based on organotypic slice co-cultures of the nigrostriatal dopaminergic system in combination with biocytin tracing and tyrosine hydroxylase labeling and subsequent automated image quantification. Selected phosphodiesterase inhibitors (PDE-Is) were studied to identify their potential growth-promoting capacities. Immunohistochemical methods were used to visualize developing fibers in the border region between ventral tegmental area/substantia nigra co-cultivated with the striatum as well as the cellular expression of PDE2A and PDE10. The quantification shows a significant increase of fiber density in the border region induced by PDE2-Is (BAY60-7550; ND7001), comparable with the potential of the nerve growth factor and in contrast to PDE10-I (MP-10). Analysis of tyrosine hydroxylase-positive fibers indicated a significant increase after treatment with BAY60-7550 and nerve growth factor in relation to dimethyl sulfoxide. Additionally, a dose-dependent increase of intracellular cGMP levels in response to the applied PDE2-Is in PDE2-transfected HEK293 cells was found. In summary, our findings show that PDE2-Is are able to significantly promote axonal outgrowth in organotypic slice co-cultures, which are a suitable model to assess growth-related effects in neuro(re)generation.

P2Y1 receptor mediated neuronal fibre outgrowth in organotypic brain slice co-cultures

“…Nevertheless, the correlation between ADPbS-and ACSFtreatment was comparable in both methods (biocytin-tracing and TH-immunolabelling). These results are in line with own previous data showing that substance-induced changes of the fibre density in the border revealed by biocytin-tracing correspond to the amount of outgrowing TH-positive fibres (Heine et al, 2013).…”

“…(ii) Automated image analysis method: The quantification of the fibre density was performed in an automated manner according to a previously described technique (Heine et al, 2013). After pre-processing and image binarization (for details see Fig.…”

“…To study these questions in an in vivo close system, organotypic slice co-cultures consisting of at least two brain slices of different parts of the same projection system are well established models. They retain the morphological characteristics of the cells and maintain the cytoarchitecture and the microenvironment of the original tissue (Cho et al, 2007;Del Rio and Soriano, 2010;Heine et al, 2013;Stavridis et al, 2009). This threedimensional environment has tremendous value for evaluating the efficacy of compounds, by preserving external mechanical inputs, the interactions between different cell structures like neurons and glial cells and cell adhesion parameters, which all profoundly affect intracellular signalling (for details see Daviaud et al, 2013).…”

Dorsal root ganglion axon bifurcation tolerates increased cyclic GMP levels: the role of phosphodiesterase 2A and scavenger receptor Npr3

“…It is generally thought that cGMP and cAMP signaling pathways are connected in a complex network including a precise spatial organization, and cGMP regulates cAMP signaling by activating or inhibiting cAMP‐degrading PDEs (Zaccolo & Movsesian, ). Previous in vitro experiments using organotypic slice cultures or growth cone turning assays revealed a link between cAMP and cGMP signaling in growth cone extension and steering (Song et al ., ; Tojima et al ., ; Heine et al ., ). Our results show that PDE2A‐mediated cyclic nucleotide cross‐talk is not required for sensory axons to branch or to grow correctly into the spinal cord in vivo .…”

Nimodipine enhances neurite outgrowth in dopaminergic brain slice co‐cultures

“…After pre‐processing of the images (for details see Heine et al, 2013), neurite structures appeared as well‐defined bright regions. Subsequently, the image area occupied by the neurites was measured to obtain a reasonable estimate of the underlying neurite density of the analyzed specimens.…”