Quantifying Filopodia in Cultured Astrocytes by an Algorithm

Abstract

Astrocytes in vivo extend thin processes termed peripheral astrocyte processes (PAPs), in particular around synapses where they can mediate glia-neuronal communication. The relation of PAPs to synapses is not based on coincidence, but it is not clear which stimuli and mechanisms lead to their formation and are active during process extension/ retraction in response to neuronal activity. Also, the molecular basis of the extremely fine PAP morphology (often 50 to 100 nm) is not understood. These open questions can be best investigated under in vitro conditions studying glial filopodia. We have previously analyzed filopodial mechanisms (Lavialle et al. PNAS 108:12915) applying an automated method for filopodia morphometry, which is now described in greater detail. The Filopodia Specific Shape Factor (FSSF) developed integrates number and length of filopodia. It quantifies filopodia independent of overall astrocytic shape or size, which can be intricate in itself. The algorithm supplied here permits automated image processing and measurements using ImageJ. Cells have to be sampled in higher numbers to obtain significant results. We validate the FSSF, and characterize the systematic influence of thresholding and camera pixel grid on measurements. We provide exemplary results of substance-induced filopodia dynamics (glutamate, mGluR agonists, EGF), and show that filopodia formation is highly sensitive to medium pH (CO) and duration of cell culture. Although the FSSF was developed to study astrocyte filopodia with focus on the perisynaptic glial sheath, we expect that this parameter can also be applied to neuronal growth cones, non-neural cell types, or cell lines.

L-type Voltage-gated Calcium Channel Modulators Inhibit Glutamate-induced Morphology in Astrocytoma Cells

“…In this study, L-type voltage-gated calcium channels (CaV) are shown to play a significant role in the glutamate-induced filopodiagenesis of astrocyte model cells U118-MG. Of the three protein subtypes constituting L-type CaV, the α2δ subunit appears essential to control filopodia extension, while the transmembrane channel α1 subunit may only be partially involved. These results are consistent with what has been observed in primary astrocytes (Cornell-Bell, Thomas, and Caffrey 1992;Cornell-Bell, Prem, and Smith 1990;Araque et al 1999;Aumann et al 2017). In our study, sequestering the extracellular calcium during glutamate stimulation prevented the protrusion of cell processes; while, sequestering the intracellular calcium still elicited about a third of the response (Fig.…”

Design of an imaging probe to monitor real-time redistribution of L-type voltage gated calcium channels in astrocytic glutamate signalling

“…Imaging began 10 minute before stimulating cells by adding a glutamate (Alfa Aesar A15031) aliquot to obtain a final dish concentration of 100 μM glutamate. More specifically, a 20x stock solution of L-glutamate was prepared in deionized millipore water and filter-sterilized through a 0.22 μm syringe filter [15, 17]. A 10.0 μL aliquot of the 20x glutamate solution was carefully delivered to the medium (2.0 mL) at the edge of the 35 mm dish.…”

Cytonemes, Their Formation, Regulation, and Roles in Signaling and Communication in Tumorigenesis

“…In addition, a novel mechanism of cell to cell communication based on cellular protrusions (filopodia) has been described in glial cells [104]. Astrocytes in vivo extend thin processes around synapses that mediate the communication with neurons.…”

“…Astrocytes in vivo extend thin processes around synapses that mediate the communication with neurons. These structures are known as peripheral astrocyte processes (PAPs) and are from 50 to 100 nm thick [104].…”