Journal: The Journal of Neuroscience

Article Title: NMDA Receptors Increase the Size of GABAergic Terminals and Enhance GABA Release

doi: 10.1523/JNEUROSCI.4980-04.2005

Figure Lengend Snippet: Enlarged GABAergic varicosities are active release units that express synaptic markers. A, Enlarged GABAergic terminals express synapsin1 and VAMP2 and accumulate the styril dye FM4-64. Left, Representative images of cultures from GAD65-eGFP mice at DIV8 grown in the absence (control) or the presence of 100 μm NMDA (NMDA). Samples were fixed and immunostained with antibodies against synapsin1 or VAMP2 or loaded with the styril dye FM4-64. eGFP (green), synapsin1, VAMP2, or FM4-64 (red) and the colocalization is shown in overlapped insets at the bottom. Right, Summary data on the analyses of colocalized eGFP with synapsin, VAMP2, and FM4-64 showing the percentage of positive GABAergic terminals for the different probes and the averaged and integrated fluorescence intensity for each probe in a given varicosity. Data are derived from at least 21 segments in each experimental group from three separate cultures. *p < 0.01, statistical significance compared with controls. B, Lack of effect of NMDA on the size of glutamatergic terminals. Left, vGlut-1 and vGlut-2 immunostaining in cultured cerebellar neurons grown for 8 d in control conditions or treated with 100 μm NMDA. Right, Summary results of the effect of NMDA on glutamatergic terminals. The area of each cluster, cluster density, and averaged and integrated intensity are expressed as percentage of untreated samples. Data derive from 13 segments from two separate cultures. Scale bars: A, B, 8 μm.

Article Snippet: Cells were then incubated for 1 hr with one of the following primary antibodies: GAD65 monoclonal antibody (GAD6; 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit anti-synapsin1 antibody (1:1000; Chemicon, Temecula, CA), rabbit anit-VAMP2 antibody (1:1000; Alomone Laboratories), and mouse anti-vGlut-1 (0.5 μg/ml) and anti-vGlut-2 (1 μg/ml; Synaptic System, Goettingen, Germany).

Techniques: Fluorescence, Derivative Assay, Immunostaining, Cell Culture