Microdissection and culturing of adult lateral entorhinal cortex layer II neurons from APP/PS1 Alzheimer model mice

Abstract

Background: Primary neuronal cultures enable cell-biological studies of Alzheimer's disease (AD), albeit typically non-neuron-specific. The first cortical neurons affected in AD reside in layer II of the lateralmost part of the entorhinal cortex, and they undergo early accumulation of intracellular amyloid-β, form subsequent tau pathology, and start degenerating pre-symptomatically. These vulnerable entorhinal neurons uniquely express the glycoprotein reelin and provide selective inputs to the hippocampal memory system. Gaining a more direct access to study these neurons is therefore highly relevant. New method: We demonstrate a methodological approach for microdissection and long-term culturing of adult lateral entorhinal layer II-neurons from AD-model mice. Results: We maintain adult microdissected lateralmost entorhinal layer II-neurons beyond two months in culture. We show that they express neuronal markers, and that they are electrophysiologically active by 15 days in vitro and continuing beyond 2 months. Comparison with existing methods: Primary neurons are typically harvested from embryonic or early postnatal brains because such neurons are easier to culture compared to adult neurons. Methods to culture adult primary neurons have been reported, however, to our knowledge, culturing of adult entorhinal subregion-specific primary neurons from AD-model animals has not been reported. Conclusions: Our methodological approach offers a window to study initial pathological changes in the AD disease-cascade. This includes the study of proteinopathy, single-neuron changes, and network-level dysfunction.

Reverse Engineering of Feedforward Cortical-Hippocampal Neural Networks Relevant for Preclinical Disease Modelling

“…To further demonstrate the wider applicability of our model system for reverse engineering anatomically relevant networks for preclinical disease modelling, we used this system to culture neurons extracted from brain regions of interest from adult AD model animals. This method for the dissection and culturing of layer specific entorhinal and hippocampal neurons from AD model rats and mice builds upon our recently published protocol for extraction and culturing of LEC LII neurons from adult AD model APP/PS1 mice (39). We found that adult neurons from both AD model rats-(Figure 5) and mice (Figure S2) were able to re-form structural connections in vitro.…”

“…Our neural cultures comprised either commercially available rat embryonic cortical and hippocampal neurons (hereafter referred to as cortical-hippocampal networks), or lateral entorhinal cortex layer II neurons (LEC-LII), Dentate Gyrus granular neurons (DG-gl), CA3 pyramidal neurons (CA3-pyr) and CA1 pyramidal neurons (CA1pyr), freshly dissected from adult McGill-R-Thy1-APP ADmodel rats or APP/PS1 AD-model mice (hereafter referred to as adult entorhinal-hippocampal AD networks). The method for the dissection and culturing of adult hippocampal neurons was refined from our previously reported protocol for adult lateral-most LEC LII (lLEC-LII) neurons from APP/PS1 model mice (39). For all cultures, coating and establishment of an astrocytic feeder layer was conducted using the same protocol.…”

“…This causes increased levels of Aβ leading to formation of extracellular cortical Aβ-deposits starting already from around 6 weeks of age (66). The APP/PS1 mice were genotyped using a KAPA-kit as described in Hanssen et al (39). The McGill-R-Thy1-APP rat model is a transgenic familial AD model with a Wistar (HsdBrl:WH) genetic background, co-expressing mutated hAPP (APP K670M671delinsNL) and (APP V717F) (67).…”

“…Subsequently, dissection and sectioning of the brain were conducted as described previously for both mice and rats. See Hanssen et al for a detailed description of the procedure (39). Brains were extracted in a petri dish filled with Hanks Balanced Salt Solution (Thermo Fischer Scientific, 88284) kept on ice.…”

“…Thus, this network is of high interest to study at preclinical stages of AD. We and others have demonstrated that adult entorhinal and hippocampal subregion specific cells from AD model mice are able to reconnect in culture (37)(38)(39). We also recently reported a method for long-term (>2 months) culturing and recording of lateral entorhinal cortex layer II (LEC LII) neurons from AD model animals in vitro (39).…”