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In JoVE (1)
Other Publications (2)
Articles by Brianne L. Sturt in JoVE
JoVE Neuroscience
Automated Quantification of Synaptic Fluorescence in C. elegans
Department of Biological Sciences, University of Toledo
The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.
Other articles by Brianne L. Sturt on PubMed
Multiple Roles for the First Transmembrane Domain of GABAA Receptor Subunits in Neurosteroid Modulation and Spontaneous Channel Activity
Neurosteroids exert potent physiological effects by allosterically modulating synaptic and extrasynaptic GABA(A) receptors. Some endogenous neurosteroids, such as 3alpha, 21-dihydroxy-5beta-pregnan-20-one (5alpha, 3alpha-THDOC), potentiate GABA(A) receptor function by interacting with a binding pocket defined by conserved residues in the first and fourth transmembrane (TM) domains of alpha subunits. Others, such as pregnenolone sulfate (PS), inhibit GABA(A) receptor function through as-yet unidentified binding sites. Here we investigate the mechanisms of PS inhibition of mammalian GABA(A) receptors, based on studies of PS inhibition of the UNC-49 GABA receptor, a GABA(A)-like receptor from Caenorhabditis elegans. In UNC-49, a 19 residue segment of TM1 can be mutated to increase or decrease PS sensitivity over a 20-fold range. Surprisingly, substituting these UNC-49 sequences into mammalian alpha(1), beta(2), and gamma(2) subunits did not produce the corresponding effects on PS sensitivity of the resulting chimeric receptors. Therefore, it is unlikely that a conserved PS binding pocket is formed at this site. However we observed several interesting unexpected effects. First, chimeric gamma2 subunits caused increased efficacy of 5alpha, 3alpha-THDOC potentiation; second, spontaneous gating of alpha(6)beta(2)delta receptors was blocked by PS, and reduced by chimeric beta(2) subunits; and third, direct activation of alpha(6)beta(2)delta receptors by 5alpha, 3alpha-THDOC was reduced by chimeric beta(2) subunits. These results reveal novel roles for non-alpha subunits in neurosteroid modulation and direct activation, and show that the beta subunit TM1 domain is important for spontaneous activity of extrasynaptic GABA(A) receptors.
Regulated Lysosomal Trafficking As a Mechanism for Regulating GABAA Receptor Abundance at Synapses in Caenorhabditis Elegans
GABA(A) receptor plasticity is important for both normal brain function and disease progression. We are studying GABA(A) receptor plasticity in Caenorhabditis elegans using a genetic approach. Acute exposure of worms to the GABA(A) agonist muscimol hyperpolarizes postsynaptic cells, causing paralysis. Worms adapt after several hours, but show uncoordinated locomotion consistent with decreased GABA signaling. Using patch-clamp and immunofluorescence approaches, we show that GABA(A) receptors are selectively removed from synapses during adaptation. Subunit mRNA levels were unchanged, suggesting a post-transcriptional mechanism. Mutants with defective lysosome function (cup-5) show elevated GABA(A) receptor levels at synapses prior to muscimol exposure. During adaptation, these receptors are removed more slowly, and accumulate in intracellular organelles positive for the late endosome marker GFP-RAB-7. These findings suggest that chronic agonist exposure increases endocytosis and lysosomal trafficking of GABA(A) receptors, leading to reduced levels of synaptic GABA(A) receptors and reduced postsynaptic GABA sensitivity.
