The Indirect Neuron-astrocyte Coculture Assay: An <em>In Vitro</em> Set-up for the Detailed Investigation of Neuron-glia Interactions

Abstract

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g., Alzheimer's disease and Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

Deletion of the Nucleotide Exchange Factor Vav3 Enhances Axonal Complexity and Synapse Formation but Tampers Activity of Hippocampal Neuronal Networks In Vitro

“…To answer the question whether the enhanced axonal elongation and branching of Vav3 −/− and Vav2 −/− /3 −/− neurons differentially influenced the formation of synapses, hippocampal neurons of wild-type, Vav2 −/− , Vav3 −/− and Vav2 −/− /3 −/− mice were co-cultured indirectly with cortical astrocytes for 14 and 21 days [29,35]. In order to analyse neuron-specific alterations caused by the lack of Vav proteins, exclusively primary cortical wild-type astrocytes were used for the co-cultures.…”

“…To analyse the synaptogenesis, hippocampal neurons and cortical astrocytes were co-cultured indirectly [29,35,73]. For the cultivation of cortical astrocytes, postnatal mice (P1-P3, SV129) were decapitated.…”

“…Therefore, we cultured hippocampal neurons lacking Vav2, Vav3 and Vav2/3 in the presence of native cortical astrocytes in a co-culture setup. This system allows the cultivation of neuronal networks in completely defined medium and the subsequent analysis of structural synapses in vitro [28][29][30][31]. In this model, we first assessed the morphological differentiation of axons and dendrites using specific markers.…”

Extracellular matrix supports excitation-inhibition balance in neuronal networks by stabilizing inhibitory synapses

“…Primary cultures of neurons and astrocytes were prepared as described previously (Gottschling et al, 2016). Hippocampal neurons were obtained at embryonic day 15 (E15) and cortical astrocytes were obtained at postnatal day 1 (P1) from male and female mice.…”

Involvement of the guanine nucleotide exchange factor Vav3 in central nervous system development and plasticity

“…In order to advance our understanding of Vav gene functions in neuronal development, we have developed a model system of embryonic hippocampal neurons in coculture with primary astrocytes in vitro that allows for the study of synapse formation over a period of 4 weeks in completely defined medium (Pyka et al, 2011a;Dzyubenko et al, 2016b;Gottschling et al, 2016a) (Figure 3A-C). The physiological relevance of the model is illustrated by the emergence of electrophysiological network activity within 7 days in vitro (Geissler and Faissner, 2012).…”