GABA has a presynaptic inhibitory effect at Lumbricus terrestris body wall muscle synapses

Abstract

Earthworm body wall muscle synapses have been suggested to contain both excitatory and inhibitory inputs, and therefore allow for investigation of excitatory/inhibitory signaling in an easily accessible model system. While previous studies have focused on postsynaptic GABAergic inhibitory mechanisms, this study investigated the hypothesis that GABAergic signaling also has presynaptic inhibitory function. This hypothesis was tested by loading synaptogreen C4 dye (also called FM1-43) into presynaptic vesicles in the presence of GABA at Lumbricus terrestris longitudinal muscle synapses. GABA treatment significantly reduced the fluorescence intensity observed at these synapses, suggesting that GABAergic signaling does indeed have a presynaptic inhibitory mechanism.

Figure 1. GABA treatment significantly reduced presynaptic synaptogreen C4 fluorescence intensity

Panel A. Representative image sets of synaptogreen C4 staining from the different treatment groups: control (no GABA treatment), top; 100 µM GABA, middle; 200 µM GABA, bottom.  Each set includes fluorescent staining only (left), and the fluorescent staining overlaid with the corresponding DIC imagefrom the same field (right). Scale bars are 20 µm.  Panel B. Scatter plot of individual data points (left) and summary data (right) for the three treatment groups.  Three earthworms were used for each treatment group, with data collected from seven to nine randomly selected confocal fields for each worm.  The number of areas analyzed for each group were n = 1504 for control (no GABA treatment); n = 1391 for 100 µM GABA; and n = 1497 for 200 µM GABA.  A Kruskal-Wallis test showed a statistically significant difference among the groups (p <0.0001).  Dunn’s multiple comparison tests revealed that 100 µM GABA and 200 µM GABA both significantly reduced the fluorescence intensity of synaptogreen C4 staining compared to the control (no GABA) group (each with p < 0.0001), but that the two GABA treatment groups were not significantly different from each other (p > 0.05).