Neurons and astrocytes are in close proximity. (A) Projection image from a stack obtained by two-photon laser-scanning microscopy in a slice of a GFAP-eGFP transgenic animal, showing eGFP-positive astrocytes in the MNTB area. Processes of a single astrocyte (arrow) are in contact with at least two principal neurons (indicated by asterisks). Arrowhead indicates eGFP-positive astrocytic endfeet surrounding a blood vessel. Bar, 20 µm. (B) Immunolabeling of a vibratome section from an eGFP mouse with GFAP antibodies coupled with Alexa 594 (red); overlay shows that most eGFP-positive cells (green) are also labeled for GFAP (yellow), although there is no complete overlap (asterisk indicates a GFAP-positive cell that is negative for eGFP). Usually the eGFP signal is more pronounced in the cell body, whereas GFAP labeling is more prominent in processes. The principal neurons appear as hollow dark circles (white arrowheads). Note that the amount of GFAP is much higher in close vicinity to the meninges (bottom right corner). Bar, 20 µm. (C) Overview of an MNTB principal neuron (PN) surrounded by black processes of astrocytes (arrows). (D) Magnified image of outlined area in C. Astrocytes extend their fine processes between the fingers of the calyx of Held (CoH) and the principal neuron (PN). (E and F) Further examples that show how astrocytic processes interdigitate between the fingers of the calyx (CoH) and principal neuron (PN). Black arrowheads indicate active zones (AZ) between a principal neuron and a CoH. Puncta adherentia (PA; white arrowhead) are shown in E. The astrocytic compartments in close vicinity to the calyx contain mitochondria (mit, in F). Note the close apposition of the labeled glial process (white arrow) to the active zone. Bars: (C) 2 µm; (D–F) 0.2 µm.