Brain Region-Specific Neuronal Networks in vitro

In the last three decades there has been a steady increase in studies using neuronal cells, which have been successfully grown in vitro as monolayer cultures. In such cultures, dissociated cells grow on a flat surface, extend processes, form synapses, and create neuronal networks. In vitro neuronal monolayer cultures are increasingly recognised as tools to study and characterize the functional response of neuronal networks to substances, bridging the gap between high-throughput approaches, genomics, and proteomics on the one side and animal models on the other.

Neuronal networks in vitro are small enough to be completely accessible for thorough studies. Simultaneously they are systems which are complex enough to improve the predictability of drug effects in the tissue of origin. Neuronal networks cultured under our conditions remain tissue-specific and maintain the pharmacologically histiotypic receptor compositions of their parent tissues. We have studied substances acting on the excitatory, inhibitory and modulatory receptor systems; and we are able to maintain information for physiological relevant mode of actions of compounds acting at the Glutamate (NMDA, AMPA, Kainate), Acetylcholine (nicotinic and muscarinic), Serotonin, Dopamine, Noradrenalin, GABA-A, GABA-B, Glycine and various other receptor systems.

Unlike systems with neuronal stem cells or progenitor cells, primary cultures develop from a mixture of different types of neurons and glial cells. The glial cells have important auxiliary functions for metabolism, and for the supplying of neurons with ions and nutrients. For years, glial cells have been relegated to a mere passive neuron-supportive element of the synapse. However, over the last decade a number of studies have conclusively shown that the glia is a necessary participant in the dialogue that neurons carry out at the synapse. Glial cells behave like additional targets of neuronal signals and respond to the synaptic release of neurotransmitters and neuromodulators. The concept of tripart synapses (pre-, postsynapse and glia) has been accepted to explain the contribution of glia in the synaptic transmission and information processing in the CNS. Neurons and glial cells share extracellular matrix elements produced by both neurons and astrocytes. Glial cells play important roles in interactions, neuron activity, growth, and axonal guidance.

Brain-region specific cell culture systems are well established for spinal cord and frontal cortex. The tissues are harvested from NMRI mice betwenn embryonic day 14 and 17.5, depending on the brain region. After ethyl ether anesthesia, the mice are euthanized by cervical dislocation according to the German Animal Protection Act. The various cell cultures for specific brain regions arel than be dissociated, seeded and grown according to established cell culture procedures in our lab. The co-culture of neurons with glial cells ensures the long-term stability after the brain region-characteristic and stable signal patterns of electrical activity have developed.

Activity starts after approximately three to four days in vitro in the form of random spiking, which, after 4 weeks in culture, stabilises into a synchronised activity pattern composed of one coordinated main burst pattern (frontal cortex); or one coordinated main burst pattern and several coordinated sub patterns (dependent on the burst strength) with interburst spiking (spinal cord). Such networks remain spontaneously active and pharmacologically responsive for more than six months and show a high intercultural reproducibility. This allows that both acute and long-term treatments can be performed. The absence of a blood brain barrier in this experimental system reflects the leaky barriers of an inflammation situation.