Establishing an Adult Mouse Brain Hippocampal Organotypic Slice Culture System that Allows for Tracing and Pharmacological Manipulation of ex vivo Neurogenesis

Background

The hippocampus is a unique region of the brain with a high degree of plasticity as a result of the ongoing neurogenesis in the dentate gyrus throughout life. This process of adult hippocampal neurogenesis starts with the asymmetric division of neural stem cells (NSCs) in the subgranular zone (SGZ) that preserves the stem cell pool and generates progenitor cells poised for neuronal differentiation (Kempermann et al., 2004; Anacker and Hen, 2017; Toda and Gage, 2018). These latter cells go through a well-defined sequence of distinct stages: NSC-generated transiently amplifying progenitor cells divide rapidly and give rise to neuroblasts. Neuroblasts are characterized by their expression of the immature neuronal marker Doublecortin (Dcx). They exit the cell cycle to differentiate into immature post-mitotic neurons that in addition to Dcx transiently express the Calcium-binding protein Calretinin as well as the neuronal marker NeuN. In the final step, these newly generated neurons migrate and integrate functionally into the existing neuronal network as mature granule cell neurons which cease to express Dcx and Calretinin, but maintain NeuN expression.

Adult hippocampal neurogenesis plays a fundamental role in physiological CNS functions such as memory consolidation and cognitive flexibility. Thus, not surprisingly, it is implicated in pathologies like Alzheimer’s disease, depression, or schizophrenia (Winner and Winkler, 2015; Anacker and Hen, 2017; Moreno-Jimenez et al., 2019; Park, 2019) and therefore represents an important target for pharmaceutical intervention. However, drug testing is currently often based on cell culture systems that lack the complex architecture and connectivity of the intact brain (Pena, 2010; Humpel, 2015). On the other hand, drug testing in the intact brains of hundreds of animals is not practicable. Organotypic brain slices thus constitute a potential intermediate step as they maintain higher brain cyto-architecture and, as multiple sections can be obtained from one brain, enable comparison of different time points and/or drug concentrations with greatly reduced inter-animal variability. To study the effects of these drugs on neurogenesis, however, a reliable and easy lineage tracing approach is required.

Previously, Kim and colleagues established a protocol to culture adult mouse organotypic hippocampal slices for an extended period of four weeks by employing a serum-free culture medium (Kim et al., 2013). Moreover, they introduced a Hibernate-A based dissection medium as an alternative to more complicated media that required constant gassing with CO₂. However, they were unable to trace the fate of single cells, nor did their slices maintain the essential afferent connection to the entorhinal cortex. Kleine Borgmann and co-workers used a different sectioning approach to overcome the latter problem and employed retrovirus labelling to study lineage progression in the SGZ (Kleine Borgmann et al., 2013). Here, we combined and refined these existing organotypic adult slice culture protocols. Moreover, we established EdU label tracing as a suitable read-out for neurogenesis. To circumvent the problem of a decreased neurogenic efficiency following a prolonged culture period, we utilized the Cyclooxygenase-inhibitor Indomethacin that has been shown before to reduce gliogenesis and exert protective effects on neurogenesis in vivo and ex vivo (Gerlach et al., 2016; Hain et al., 2018; Melo-Salas et al., 2018). Finally, establishing proof of concept as to the value of this protocol we compared the effects of Silychristin, iopanoic acid, and BCH on neurogenesis. These drugs are small molecular inhibitors of the monocarboxylate transporter 8 (Mct8), deiodinase type 2 (Dio2), and L-type amino acid transporters 1/2 (Lat 1/2) respectively–components of central thyroid hormone signalling. While neither iopanoic acid nor BCH treatment altered ex vivo neurogenesis, the number of new neurons was significantly reduced in Silychristin-treated cultures (Mayerl et al., 2020).

Taken together, these improvements enabled us to establish a method that allows for i) an extended culture period of adult brain slices for at least three weeks with good neurogenic efficiency, ii) easy lineage tracing using EdU that also provides a defined and controlled starting point, and iii) pharmacological manipulation of ex vivo hippocampal neurogenesis. We believe that our advanced methodology will be useful for future drug screening approaches to sustain or improve hippocampal neurogenesis in various pathological conditions or physiological alterations such as ageing.