Refined protocol for quantifying hippocampal neurogenesis in organotypic slice cultures.

While human iPSC-derived models of the brain provide valuable genetic context, organotypic slice cultures from rodents remain essential for capturing the physiological complexity of intact neural tissue. This protocol describes a refinement of the interface method originally established by Stoppini and colleagues in 1991, providing a robust platform for the long-term cultivation of rodent hippocampal slices. Unlike dissociated monolayer systems, these 3-dimensional (3D) brain slices preserve the endogenous cytoarchitecture and the native extracellular matrix (ECM) of the central nervous system (CNS). This structural integrity is paramount for investigating hippocampal neurogenesis, as the proliferation, migration, and circuit integration of neural progenitors are governed by mechanical and biochemical cues present in the specialized local niche. By maintaining the spatial relationships between neurons, astrocytes, and microglia, this organotypic system replicates the arrangement of hippocampal neural networks more precisely than emergent stem cell models. Our protocol outlines an optimized workflow for McIlwain tissue chopper-based sectioning and maintenance of slices on porous hydrophilic membranes, ensuring efficient nutrient exchange and sustained tissue viability for several weeks. This configuration provides simultaneous access to the hippocampal slices and culture medium, facilitating the use of multiple investigative techniques such as live-cell imaging, electrophysiology, or pharmacological treatments. Despite relying predominantly on rodent tissue due to the scarcity of live human brain samples; the ability to observe developmental processes within the preserved architectural framework of the hippocampus makes this technique a cornerstone for studying neurogenesis and circuit assembly. Importantly, this refined approach bridges the gap between disorganised monolayers of