Ca 2+ transients in astrocyte fine processes occur via Ca 2+ influx in the adult mouse hippocampus

Glia. 2016 Dec;64(12):2093-2103. doi: 10.1002/glia.23042. Epub 2016 Aug 1.

Abstract

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca2+ -sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two-photon imaging of Ca2+ transients in adult CA1 hippocampal astrocytes using a new single-cell patch-loading strategy to image Ca2+ -sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca2+ transients whereas astrocyte soma rarely showed spontaneous Ca2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L- or T-type voltage-gated calcium channels. Removal of extracellular Ca2+ almost completely and reversibly abolished the spontaneous signals while IP3 R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca2+ . The Ca2+ influx dependency of the spontaneous signals in patch-loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093-2103.

Keywords: Calcium Green-1 dextran; Fluo-4; GCaMP; IP3R2; TRPA1; calcium channel; glia; microdomain; synaptic; two-photon.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / drug effects
  • Animals
  • Astrocytes / metabolism*
  • Calcium / metabolism*
  • Calcium Channels, L-Type / genetics
  • Calcium Channels, L-Type / metabolism
  • Calcium Signaling / drug effects
  • Carbenoxolone / pharmacology
  • Chromones / pharmacology
  • Excitatory Amino Acid Antagonists / pharmacology
  • Excitatory Amino Acid Transporter 1 / genetics
  • Excitatory Amino Acid Transporter 1 / metabolism
  • Female
  • Hippocampus / cytology*
  • In Vitro Techniques
  • Inositol 1,4,5-Trisphosphate Receptors / genetics
  • Inositol 1,4,5-Trisphosphate Receptors / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Male
  • Membrane Microdomains / drug effects
  • Membrane Microdomains / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Pyridines / pharmacology
  • TRPA1 Cation Channel / genetics
  • TRPA1 Cation Channel / metabolism

Substances

  • 7-(hydroxyimino)cyclopropan(b)chromen-1a-carbxoylic acid ethyl ester
  • Cacna1d protein, mouse
  • Calcium Channels, L-Type
  • Chromones
  • Excitatory Amino Acid Antagonists
  • Excitatory Amino Acid Transporter 1
  • Inositol 1,4,5-Trisphosphate Receptors
  • Ip3r2 protein, mouse
  • Luminescent Proteins
  • Pyridines
  • Slc1a3 protein, mouse
  • TRPA1 Cation Channel
  • Trpa1 protein, mouse
  • 6-methyl-2-(phenylethynyl)pyridine
  • Carbenoxolone
  • Calcium