The brain is a fascinating structure to investigate not only because of its staggering complexity but also because of its clinical importance. The human brain consists of more than 80 billion nerve cells, each receiving up to 10000 input connections. Despite big efforts in neuroscience, we still do not understand how the brain carries out its basic functions, such as information processing and storage, because the critical link between network structure and function remains largely unclear. The bottom-up neuroscience approach aims to answer these questions by engineering small, patterned networks of neurons in vitro. The complexity of the investigated system is thereby drastically reduced compared to top-down neuroscience approach, which examines the immensely complex brain as a whole.
Advances in neuroscience have been driven by the development of new tools, such as the light microscope, patch-clamp, or more recently optogenetics. Here, the investigation of small neuronal networks is improved by developing new tools for culturing, measuring and stimulating neurons.
First, the survival and function of neurons cultured in vitro at low densities are improved by suspending astrocytes cultured on cellulose-paper above the neurons. Astrocytes are a critical cell type in the brain and their addition in vitro creates a supportive and more physiological micro-environment, in particular for networks of low densities typically needed for bottom-up neuroscience. Astrocytic co-culture results in drastically improved neuronal viability and more frequent spontaneous spiking activity compared to mono-cultures.
Second, the analysis of neuronal calcium imaging videos is simplified with an easy-to-use and robust computer program. Calcium imaging is a convenient way to measure the electrophysiological activity of neurons using fluorescence microscopy. Extracting the neuronal activity from these videos however is challenging due to the enormous amounts of data it generates. The developed tool allows to efficiently perform this repetitive task and frees up time for the actual analysis of the behavior of the recorded network of neurons.
Third, the stimulation of neurons in vitro is improved with a local chemical stimulation platform based on the FluidFM, a force-controlled nanopipette. After gently approaching the FluidFM cantilever to the target neuron, the neurotransmitter glutamate is dispensed locally to stimulate the cell, thereby replicating the signal transmission between neurons. This platform is capable of reliably stimulating neurons with a control over the stimulation dose as can be measured electrically and optically on the level of both the stimulated neuron and the network.
Finally, because the brain uses several different kinds of neurotransmitters and neuromodulators, it is advantageous to extend the FluidFM to contain multiple channels to allow it to release multiple compounds during an experiment. The required instrumentation for interfacing such novel cantilevers was developed and tested with prototypes for future developments.
Overall, these techniques expand the toolkit for engineering and investigating networks of neurons and to improve our understanding of the brain
