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We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month.

In the current global climate change scenario, stressors overlap in space and time, and knowledge on the effects of their interaction is highly needed to understand and predict the response and resilience of organisms. Corals, among many other benthic organisms, are affected by an increasing number of global change-related stressors including warming and invasive species. In this study, the cumulative effects between warming and invasive algae were experimentally assessed on the temperate reef-builder coral Cladocora caespitosa. We first investigated the potential local adaptation to thermal stress in two distant populations subjected to contrasting thermal and necrosis histories. No significant differences were found between populations. Colonies from both populations suffered no necrosis after long-term exposure to temperatures up to 29 °C. Second, we tested the effects of the interaction of both warming and the presence of invasive algae. The combined exposure triggered critical synergistic effects on photosynthetic efficiency and tissue necrosis. At the end of the experiment, over 90% of the colonies subjected to warming and invasive algae showed signs of necrosis. The results are of particular concern when considering the predicted increase of extreme climatic events and the spread of invasive species in the Mediterranean and other seas in the future.