Re-innervation of the Denervated Dentate Gyrus by Sprouting Associational and Commissural Mossy Cell Axons in Organotypic Tissue Cultures of Entorhinal Cortex and Hippocampus.

Domenico Del Turco,Lars Hildebrandt-Einfeldt,Viktor J Beeg Moreno,Thomas Deller,Mandy H Paul

doi:10.3389/fnmol.2019.00270

Domenico Del Turco, Lars Hildebrandt-Einfeldt + Show 3 more

Open Access

https://doi.org/10.3389/fnmol.2019.00270

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Abstract

Collateral sprouting of surviving axons contributes to the synaptic reorganization after brain injury. To study this clinically relevant phenomenon, we used complex organotypic tissue cultures of mouse entorhinal cortex (EC) and hippocampus (H). Single EC-H cultures were generated to analyze associational sprouting, and double EC-H cultures were used to evaluate commissural sprouting of mossy cells in the dentate gyrus (DG) following entorhinal denervation. Entorhinal denervation (transection of the perforant path) was performed at 14 days in vitro (DIV) and associational/commissural sprouting was assessed at 28 DIV. First, associational sprouting was studied in genetically hybrid EC-H cultures of beta-actin-GFPtg and wild-type mice. Using calretinin as a marker, associational axons were found to re-innervate almost the entire entorhinal target zone. Denervation experiments performed with EC-H cultures of Thy1-YFPtg mice, in which mossy cells are YFP-positive, confirmed that the overwhelming majority of sprouting associational calretinin-positive axons are mossy cell axons. Second, we analyzed associational/commissural sprouting by combining wild-type EC-H cultures with calretinin-deficient EC-H cultures. In these cultures, only wild-type mossy cells contain calretinin, and associational and commissural mossy cell collaterals can be distinguished using calretinin as a marker. Nearly the entire DG entorhinal target zone was re-innervated by sprouting of associational and commissural mossy cell axons. Finally, viral labeling of newly formed associational/commissural axons revealed a rapid post-lesional sprouting response. These findings demonstrate extensive and rapid re-innervation of the denervated DG outer molecular layer by associational and commissural mossy cell axons, similar to what has been reported to occur in juvenile rodent DG in vivo.

Highlights

Regardless of the underlying cause, central nervous system (CNS) injuries result in primary damage at the lesion site and secondary denervation damage in connected brain areas
To determine whether the laminar termination pattern of the two major projection systems to the dentate gyrus, i.e., the calretininpositive associational projection arising from hilar mossy cells and the entorhinal projection arising from layer II neurons of the entorhinal cortex (EC), were maintained in organotypic tissue cultures, genetically hybrid cultures were generated at postnatal day 4–5
A segregated termination of calretinin-positive associational and entorhinal axons could be seen at all levels of the re-sliced dentate gyrus (DG) (Figure 1), the most defined termination pattern of the two projections was found in middle sections (Figures 1b,c)

Summary

Introduction

Regardless of the underlying cause, central nervous system (CNS) injuries result in primary damage at the lesion site and secondary denervation damage in connected brain areas. Even spatially circumscribed CNS lesions cannot be considered local injuries since every lesion challenges the neuronal network In line with this view of CNS damage, the CNS reacts to lesions with a profound rewiring of its connections. This lesion-induced plastic response may homeostatically restabilize or destabilize the perturbed network, causing changes in the throughput of information in denervated areas (Steward, 1994; Vlachos et al, 2012a,b, 2013). One of the mechanisms contributing to the reorganization of connections in denervated areas of the CNS is collateral sprouting of surviving axons. A reinvestigation of the mechanisms underlying denervationinduced collateral sprouting may be useful in the search for novel targets

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