Organotypic brain culture is an experimental tool widely used in neuroscience studies. One major
drawback of this technique is reduced neuronal survival across time, which is likely exacerbated by the
loss of blood flow. We have designed a novel, tube flow system, which is easily incorporated into the
commonly-used, standard semi-permeable membrane culture methodology which has significantly
enhanced neuronal survival in a brain stem nucleus involved in control of motivated and arousal states:
the laterodorsal tegmental nucleus (LDT). Our automated system provides nutrients and removes
waste in a comparatively aseptic environment, while preserving temperature, and oxygen levels. Using
immunohistochemistry and electrophysiology, our system was found superior to standard techniques
in preserving tissue quality and survival of LDT cells for up to 2 weeks. In summary, we provide
evidence for the first time that the LDT can be preserved in organotypic slice culture, and further, our
technical improvements of adding a flow system, which likely enhanced perfusion to the slice, were
associated with enhanced neuronal survival. Our perfusion system is expected to facilitate organotypic
experiments focused on chronic stimulations and multielectrode recordings in the LDT, as well as
enhance neuronal survival in slice cultures originating from other brain regions.