FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes

Abstract

The amphiphilic fluorescent styryl pyridinium dyes FM1-43 and FM4-64 are used to probe activity-dependent synaptic vesicle cycling in neurons. Cultured astrocytes can internalize FM1-43 and FM4-64 inside vesicles but their uptake is insensitive to the elevation of cytosolic calcium (Ca 2؉ ) concentration and the underlying mechanism remains unclear. Here we used total internal reflection fluorescence microscopy and pharmacological tools to study the mechanisms of FM4-64 uptake into cultured astrocytes from mouse neocortex. Our data show that: (i) endocytosis is not a major route for FM4-64 uptake into astrocytes; (ii) FM4-64 enters astrocytes through an aqueous pore and strongly affects Ca 2؉ homeostasis; (iii) partitioning of FM4-64 into the outer leaflet of the plasma membrane results in a facilitation of store-operated Ca 2؉ entry (SOCE) channel gating; (iv) FM4-64 permeates and competes with Ca 2؉ for entry through a SOCE channel; (v) intracellular FM4-64 mobilizes Ca 2؉ from the endoplasmic reticulum stores, conveying a positive feedback to activate SOCE and to sustain dye uptake into astrocytes. Our study demonstrates that FM dyes are not markers of cycling vesicles in astrocytes and calls for a careful interpretation of FM fluorescence.calcium homeostasis ͉ endoplasmic reticulum ͉ lipid bilayer ͉ styryl dye ͉ TIRF I n astrocytes the strong expression of the plasma membrane (PM) store-operated Ca 2ϩ entry (SOCE) (1-4) contrasts with a relatively small expression of voltage-gated channels and AMPA/ NMDA ligand-gated channels that are mostly expressed by neurons. In response to neuronal activity and neurotransmitter release, the activation of astroglial metabotropic receptors induces a reduction of the endoplasmic reticulum (ER) Ca 2ϩ concentration ([Ca 2ϩ ] ER ) that leads to the activation of the SOCE and to an elevation of the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) (4) that is critical for the electrically silent astrocytes to release gliotransmitters and control nearby neurons, glia, and blood vessels (5). The type 1-2 stromal interaction molecules (STIM1-2) and Orai1-3 membrane proteins are key components of the SOCE, being the ER Ca 2ϩ sensor and the PM pore-forming subunit, respectively (6, 7). A possible participation of the classical transient receptor potential (TRPC) channels to the protein complex generating the SOCE is debated in several cell types (8), including astrocytes (3).Styryl pyridinium FM dyes are amphiphilic molecules that reversibly partition in lipid membranes. Their positively charged pyridinium head prevents them from diffusing through the PM. Only weakly fluorescent in aqueous solution their quantum yield increases in a lipid environment. Their activity-dependent uptake and destaining has made them common probes for synaptic vesicle cycling at the nerve terminals (9-11). Cultured astrocytes internalize FM dyes (3,(12)(13)(14)(15)(16)(17)(18). However, this uptake is not modified by the activation of metabotropic glutamate receptors (19), and its mechanism remains elusiv...

APP+, a Fluorescent Analogue of the Neurotoxin MPP+, Is a Marker of Catecholamine Neurons in Brain Tissue, but Not a Fluorescent False Neurotransmitter

“…It was reported that the endocytic dye FM4-64, a pyridinium analogue of considerably larger size than APP+, is taken up by cortical astrocytes via store-operated Ca 2+ entry (SOCE) channels. 49 It was also suggested that background staining in brain slice observed with FM4-64 might be due to astrocytic uptake occurring via the SOCE channels. Organic cation transporters such as OCT3, which are widely expressed in the brain and have been shown to transport MPP+, 50,51 may also contribute to extracatecholaminergic staining by APP+.…”

A protein synthesis and nitric oxide-dependent presynaptic enhancement in persistent forms of long-term potentiation

“…A single, stable population of terminals before and after LTP It is important to verify that sampled puncta were primarily representative of CA3 terminals, and did not include large numbers of GABAergic inhibitory interneurons or astrocytes, both of which endocytose FM 1 -43 (Hablitz et al 2009;Li et al 2009) and are frequently overlooked in hippocampal FM experiments. If this were the case, one might expect different populations of destaining kinetics to be apparent in our data, as both inhibitory interneurons and astrocytes exhibit heterogeneity in their release properties (McBain and Fisahn 2001;Moulder et al 2007;Sakaba 2008).…”

Fluorescent AM1-43 and FM1-43 probes for dental sensory nerves and cells: Their labeling mechanisms and applications

“…been reported to label hair cells of lateral line organs [2,[11][12][13][14][15][16][17][18][19][20] and the inner ear [19,[21][22][23][24][25][26][27][28][29]. Other sensory cells and nerves are also labeled with FM1-43, AM1-43 and related dyes: vibrissal follicles, palatal rugae, enteric neurons, nasal mucosa, Merkel cells, muscle spindles, cultured astrocytes, sensory neurons of trigeminal and dorsal root ganglia, sensory receptors of visceral pleura, and cough receptors of vagal sensory neurons [22,[30][31][32][33][34][35][36][37][38][39][40].…”

“…Although astrocytes are not sensory neurons, the FM1-43-related dye FM4-64, enters a store-operated calcium channel of cultured astrocytes [37]. Karashima et al [61] reported on the other channel of transient receptor potential (TRP) family that TRPA1-transfected Chinese hamster ovary (CHO) cells are labeled with FM1-43 following stimulation with a TRPA1 agonist.…”