Excess extracellular acetylcholinesterase (AChE) decreases glutamatergic synapses. A. Confocal microscopic images showing immunofluorescent staining of synapsin in cultured hippocampal neurons at the 12th day in vitro (DIV). Panels show, from left to right, neurons in control medium, in synaptic AChE (AChE-S)-conditioned medium containing 10 μM physostigmine (AChE + physo.) and in control medium containing physostigmine (physo. 10 μM). White bar represents 20 μm. B. Numbers of synapsin-positive clusters per image field in neurons grown under various conditions (control: 392 ± 26 clusters/field, n = 12 images; AChE + physo: 141 ± 33 clusters/field, n = 15 images; Physo. 381 ± 22 clusters/field, n = 9 images; * P < 0.05). C. Immunofluorescent staining of the GluR2 subunit of the glutamate receptor in cultured hippocampal neurons at the 12th DIV. Rows represent neurons gown under various culture conditions: top row: control medium; middle row: AChE-conditioned medium + 10 μM physostigmine; bottom row: physostigmine alone. The GluR2 subunits on the membrane surface were stained first under non-permeabilizing conditions (red, middle column) and the intracellular GluR2 subunits were then stained under permeabilizing conditions (green, left column). Right column shows overlay images of surface and intracellular GluR2 subunits. C’. Enlarged images of the areas enclosed by a dotted line in C, illustrating GluR2 clusters in control and AChE-treated neurons. Notably, increased extracellular AChE reduced the number of surface GluR2 subunit clusters but did not affect the immunoreactivity of intracellular GluR2 subunits. D. Numbers of surface GluR2-positive clusters per image field in neurons cultured in various media (control: 440 ± 28 clusters/field, n = 8 images; AChE + physo.: 256 ± 50 clusters/field, n = 12 images; Physo.: 399 ± 48 clusters/field, n = 8 images; * P < 0.05).