NT2 Derived Neuronal and Astrocytic Network Signalling

Abstract

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.

Research tool: validation of floxed α7 nicotinic acetylcholine receptor conditional knockout mice using in vitro and in vivo approaches

“…; Hill et al . ; Pirttimaki & Parri, ). Thus, astrocytes are probably a significant component of the molecular, synaptic and cognitive consequences of aberrant Aβ accumulation in AD pathogenesis that have largely been overlooked (Teaktong et al .…”

“…We and others recently showed that a key mediator of astrocyte α7 nAChR function is Aβ, a ubiquitously expressed signalling peptide and putative toxic trigger for AD (Parri & Dineley, 2010;Pirttimaki et al 2013;Talantova et al 2013;Lee et al 2014). Astrocytes play a critical role in synaptic transmission, plasticity and memory formation through the release of chemical transmitters (gliotransmitters) such as glutamate (Pirttimaki et al 2011;Hill et al 2012;Pirttimaki & Parri, 2012). Thus, astrocytes are probably a significant component of the molecular, synaptic and cognitive consequences of aberrant Aβ accumulation in AD pathogenesis that have largely been overlooked (Teaktong et al 2003); these proof-of-concept studies position us to specifically address their role in AD initiation and progression in preclinical models as well as several other cell-type-specific roles (e.g.…”

Functional Astrocyte-Neuron Lactate Shuttle in a Human Stem Cell-Derived Neuronal Network

“…21 We have recently demonstrated that NT2.D1 cell-derived neuron and astrocyte (NT2.N/A) networks can communicate and so have the potential to interact with each other as observed in vivo. 22 If this model is to be applied to the investigation of human neuronal function, it is also essential that the metabolic relationship is sufficiently representative of functional reality.…”

Effects of Lithium and Valproic Acid on Gene Expression and Phenotypic Markers in an NT2 Neurosphere Model of Neural Development

“…Future studies will identify the temporal nature of these changes in order to fully understand the mechanism of action of these compounds. In addition, we will also determine the functional effects of these compounds on neurons and astrocytes derived from these cultures [39]. Following further validation with a larger range of toxins this approach may prove useful for identifying potential developmental neurotoxins as part of a battery of tests for regulatory screening processes.…”