Basal GABA Regulates GABABR Conformation and Release Probability at Single Hippocampal Synapses

Abstract

Presynaptic GABA(B) receptor (GABA(B)R) heterodimers are composed of GB(1a)/GB(2) subunits and critically influence synaptic and cognitive functions. Here, we explored local GABA(B)R activation by integrating optical tools for monitoring receptor conformation and synaptic vesicle release at individual presynaptic boutons of hippocampal neurons. Utilizing fluorescence resonance energy transfer (FRET) spectroscopy, we detected a wide range of FRET values for CFP/YFP-tagged GB(1a)/GB(2) receptors that negatively correlated with release probabilities at single synapses. High FRET of GABA(B)Rs associated with low release probability. Notably, pharmacological manipulations that either reduced or increased basal receptor activation decreased intersynapse variability of GB(1a)/GB(2) receptor conformation. Despite variability along axons, presynaptic GABA(B)R tone was dendrite specific, having a greater impact on synapses at highly innervated proximal branches. Prolonged neuronal inactivity reduced basal receptor activation, leading to homeostatic augmentation of release probability. Our findings suggest that local variations in basal GABA concentration are a major determinant of GB(1a)/GB(2) conformational variability, which contributes to heterogeneity of neurotransmitter release at hippocampal synapses.

GABA B receptor deficiency causes failure of neuronal homeostasis in hippocampal networks

“…Notably, addition of GABA B R agonist after prolonged inactivity stabilizes a closed Synt 1 conformation, suggesting reduction in local GABA levels induces an adaptive Synt 1a response. Third, in addition to the well-known role of GABA B Rs in the presynaptic Ca 2+ flux inhibition, at a rapid timescale of minutes (22,45), our work revealed the necessity for basal GABA B R activity in presynaptic adaptations of Ca 2+ transients to chronic activity perturbations at extended timescales of days. Deletion of the GB 1a PCT domain blocks presynaptic homeostatic plasticity by disrupting GABAmediated conformational changes within the presynaptic GB 1a / Ca V 2.2/Synt 1 signaling complex.…”

“…Importantly, acute application of baclofen restored Synt 1a FRET in TTX 48h -treated neurons (Fig. 4C), suggesting prolonged silencing may promote Synt 1a opening by reducing the extracellular GABA levels (22).…”

“…Homeostatic regulation of synaptic strength represents a basic mechanism of neuronal adaptation to constant changes in ongoing activity levels. Strong evidence exists on homeostatic augmentation of Pr and readily releasable pool size in response to prolong inactivity in hippocampal neurons (19,22,(34)(35)(36)(37)(38)(39)(40)(41)(42). These homeostatic adaptations are associated with modulation of presynaptic Ca 2+ flux (29) and remodeling of a large number of proteins in presynaptic cytomatrix (7).…”

“…Activity-dependent FM1-43 or FM4-64 styryl dyes have been used to estimate synaptic vesicle exocytosis and presynaptic plasticity, as described (22). The experiments were conducted at room temperature in extracellular Tyrode solution containing (in mM): NaCl, 145; KCl, 3; glucose, 15; Hepes, 10; MgCl 2 , 1.2; CaCl 2 , 1.2, with pH adjusted to 7.4 with NaOH.…”

“…Intensity-based FRET imaging was carried as described before (22,30). Donor dequenching resulting from the desensitized acceptor was measured from Cer/CFP emission (460-500 nm) before and after the acceptor (YFP) photobleaching.…”