Fig. 6. Weak mechanical stimulation induces large vesicles in astrocytes.

(A) Two-photon microscopy images of a representative astrocyte that was preloaded with Fluo-4 AM at the time indicated in B. The position of the puffer pipette is indicated (p). The first puff induced an increase in Fluo-4 fluorescence in the astrocytic processes (i–ii). A high Fluo-4 fluorescence vesicle (1) appeared after the second puff (iii) and suddenly disappeared (iv–v). (B) Fluo-4 fluorescence in the circled area (1) indicated in A. Middle trace (Pyr), whole-cell voltage-clamp recording from a nearby pyramidal neuron with apical dendrites passing through the puffed area. Bottom traces, whole-cell recording from a representative pyramidal neuron, showing that local application (Glu and arrow) of 1 μM glutamate induced inward currents (1 μM), but 0.5 μM glutamate did not (0.5 μM). (C) The frequency of SICs during the control period (Con) and after puffs (Puff). (D) The mean relative Fluo-4 fluorescence intensity from puffed processes (Proc) before and after puffing (Puff). n = 10 slices. (E) A large vesicle was developed from small vesicles. A small vesicle (arrowhead) was being merged into a large vesicle (arrow). Images were from the same astrocyte in A. (F) Fluo-4 and FM 1-43 fluorescence showed a large vesicle with Fluo-4 fluorescence (144 s, arrow). After its Fluo-4 fluorescence disappeared (fusion occurred), the large vesicle was FM 1-43-negative with a reduced size (148 s, arrow). FM 1-43 (20 μM) was added to the puffer pipette solution (ACSF). (G) The bath application of the P2Y1 receptor antagonist MRS2179 (MRS) inhibited puffing ACSF-induced generation of large vesicles.