Technical Reference #3249

3249.

Calcium-Independent Inhibitory G-Protein Signaling Induces Persistent Presynaptic Muting of Hippocampal Synapses Devon C. Crawford; Chun Yun Chang; Krzysztof L. Hyrc; and Steven Mennerick, Washington University in St. Louis, Journal of Neuroscience, 31(3249), (2011)
Link To Paper

Abstract:
Adaptive forms of synaptic plasticity that reduce excitatory synaptic transmission in response to prolonged increases in neuronal activity may prevent runaway positive feedback in neuronal circuits. In hippocampal neurons; for example; glutamatergic presynaptic terminals are selectively silenced; creating “mute” synapses; after periods of increased neuronal activity or sustained depolarization. Previous work suggests that cAMP-dependent and proteasome-dependent mechanisms participate in silencing induction by depolarization; but upstream activators are unknown. We; therefore; tested the role of calcium and G-protein signaling in silencing induction in cultured hippocampal neurons. We found that silencing induction by depolarization was not dependent on rises in intracellular calcium; from either extracellular or intracellular sources. Silencing was; however; pertussis toxin sensitive; which suggests that inhibitory G-proteins are recruited. Surprisingly; blocking four common inhibitory G-protein-coupled receptors (GPCRs) (adenosine A1 receptors; GABAB receptors; metabotropic glutamate receptors; and CB1 cannabinoid receptors) and one ionotropic receptor with metabotropic properties (kainate receptors) failed to prevent depolarization-induced silencing. Activating a subset of these GPCRs (A1 and GABAB) with agonist application induced silencing; however; which supports the hypothesis that G-protein activation is a critical step in silencing. Overall; our results suggest that depolarization activates silencing through an atypical GPCR or through receptor-independent G-protein activation. GPCR agonist-induced silencing exhibited dependence on the ubiquitin-proteasome system; as was shown previously for depolarization-induced silencing; implicating the degradation of vital synaptic proteins in silencing by GPCR activation. These data suggest that presynaptic muting in hippocampal neurons uses a G-protein-dependent but calcium-independent mechanism to depress presynaptic vesicle release.

Materials & Methods:
Calcium imaging. All calcium imaging experiments used mass cultures. For fura-2 experiments; neurons were plated on glass-bottom dishes (MatTek Corporation). Cells were loaded with fura-2 by 60 min incubation with 5 M fura-2 AM (Invitrogen) and 0.1% Pluronic F-127 (Invitrogen) in Neurobasal medium; pH 7.2; at room temperature; washed with Neurobasal medium; and incubated for another 60 min to allow for ester hydrolysis. After loading; the cells were imaged on an Eclipse TE300 inverted microscope using a 40 (1.3 numerical aperture) oil-immersion objective (Nikon). The microscope was equipped with a 75 W xenon arc lamp and a cooled CCD camera (Cooke Corp.). The fluorescence excitation was provided by a band-specific filter (340 and 380 nm; Semrock) in combination with DM400 dichroic beam splitter (Nikon). Pairs of images were collected at alternate excitation wavelengths. The images were divided by one another to yield ratio values for individual cell bodies after subtracting the matching background. Imaging was performed at room temperature; but cultures were returned to 37°C for the period between images early in the treatment and images at the end of the 4 h treatment. For this reason; the same neurons were represented at baseline and 5 min time points; but separate fields of neurons were used for 4 h time points in Figure 1. MetaFluor software (Molecular Devices) was used for image acquisition and analysis. Fluo-4 was loaded into cells by addition of Fluo-4 AM (2 M; Invitrogen) to the culture media 30 min before the end of the 4 h KCl treatment. Medium was then exchanged for extracellular recording saline containing elevated KCl (34 mM total) and 0 mM CaCl2. Cells were imaged at room temperature using an Eclipse TE2000-S inverted microscope with 40 (0.6 numerical aperture) objective (Nikon) and a cooled 12-bit CCD camera (Photometrics). Epifluorescence was provided by a metal halide lamp; and images were acquired at 1 Hz using MetaMorph software (Molecular Devices). Calcium was briefly elevated to 0.5 mM for 5–10 s after 5 s of baseline in 0 mM calcium.

Microscopic Technique
Calcium imaging, Inverted microscopy

Cell Type(s)
hippocampal neurons