Intracellular Calcium Measurements for Functional Characterization of Neuronal Phenotypes

Abstract

The central and peripheral nervous system is built by a network of many different neuronal phenotypes together with glial and other supporting cells. The repertoire of expressed receptors and secreted neurotransmitters and neuromodulators are unique for each single neuron leading to intracellular signaling cascades, many of them involving intracellular calcium signaling. Here we suggest the use of calcium signaling analysis upon specific agonist application to reliably identify neuronal phenotypes, being important not only for basic science, but also providing a reliable tool for functional characterization of cells prior to transplantation. Calcium imaging provides various cellular information including signaling amplitudes, cell localization, duration, and frequency. Microfluorimetry reveals a signal summarizing the entire population, and its use is indicated for high-throughput screening purposes.

iPSC-derived neurons as a tool for probing molecular pharmacology of antipsychotic action

“…Indeed, we found a significant increase in mRNA of glutamate receptors, including kainate (GRIK1), and NMDA receptor subunit 1 (GRIN1) following 79 and 115 days of differentiation ( Fig 3). Remarkably, the onset of GRIK1 and GRIN1 expression in human iPSC-neurons was consistent with the Allen BrainAtlas Developmental Transcriptome, which demonstrates increased expression of these receptors as early as 8 weeks post conception in human fetal tissue (Miller et al, 2014;© 2016 Allen Institute for Brain Science). In contrast, we found no significant changes in NMDA receptor subunit 2A (GRIN2A) expression at the time periods assessed, up to 115 days, which is equivalent to approximately 16 weeks post conception.…”

“…In contrast, we found no significant changes in NMDA receptor subunit 2A (GRIN2A) expression at the time periods assessed, up to 115 days, which is equivalent to approximately 16 weeks post conception. Importantly, GRIN2A expression does not increase in humans (Miller et al, 2014;© 2016 Allen Institute for Brain Science) or rodents (Liu et al, 2004) until birth. Thus, in line with previous reports (Shi et al, 2012;Brennand et al, 2015), neuronal differentiation of human iPSCs by Dual-SMAD inhibition recapitulates aspects of human brain development.…”

“…To understand how an antipsychotic such as clozapine might modulate neuronal activity, neurons were focally stimulated with glutamate, dopamine, and 5-HT2A agonist DOI, before and after incubation with clozapine or vehicle. Calcium measurements in iPSC-neurons provide detailed functional information about neuronal phenotypes following differentiation (Glaser et al, 2016). Therefore, cells were preloaded with a fluorescent calcium indicator, and changes in calcium levels were recorded to monitor neuronal activity in response to the pharmacological challenges.…”

Mycobacterium tuberculosis Sulfolipid-1 Activates Nociceptive Neurons and Induces Cough

“…In Vitro Having established that the Mtb extract alone is sufficient to induce cough, we next investigated if the Mtb extract activates nociceptive neurons in cell culture using live cell imaging of intracellular calcium dynamics. When nociceptive neurons are triggered by nociceptive agonists, they demonstrate an increase in intracellular [Ca 2+ ] (Glaser et al, 2016) that can be quantified using Ca 2+ responsive dyes (Grynkiewicz et al, 1985;Iatridou et al, 1994;Kao et al, 2010). To first determine if the Mtb extract can trigger neurons, we used the immortalized mouse embryonic dorsal root ganglion (DRG) cell line, MED17.11, which demonstrates properties of nociceptive neurons (Doran et al, 2015).…”

Single-cell approaches for molecular classification of endocrine tumors

“…Calcium imaging with detection of signal amplitude, duration, and frequency along with cell adjacency and positional data can be captured in single-cell format to generate agonist response profiles as a means to identify the specific repertoire of expressed receptors in individual living cells [50]. The ability to perform ex-vivo provocative testing is particularly suited to the study of endocrine cell signaling.…”