UC Berkeley

GABAA receptors (GABAARs) mediate fast synaptic inhibition in the mammalian brain and are essential for balancing excitation and sharpening our sensory perceptions. GABAARs are plastic and respond to changes in neuronal activity by strengthening or weakening inhibitory neurotransmission. This inhibitory plasticity responds to many of the same signals that mediate glutamatergic excitatory plasticity and works in tandem with or in contraposition to excitation to form the cellular basis of memories. GABAARs are added to or dispersed from synapses to modulate synaptic strength, and this is achieved by lateral diffusion in the plane of the membrane. By tethering GABAARs with synaptic scaffold proteins, primarily gephyrin, the inhibitory synapse can accumulate GABAARs dependent on the potency of this interaction. The scaffolds themselves are highly plastic and can agglomerate or wash away in coordination with GABAARs. An additional element of this process is the availability of extrasynaptic GABAARs able to diffuse into synapses, a supply that is tightly controlled by endo- and exocytosis of receptors from internal endosomal reservoirs and which is important for both acute plasticity and long-term homeostasis. The result is inhibitory synapses that are able to respond to activity changes with structural rearrangements on the scale of minutes to days.

Thus far, GABAAR mobility has been primarily interrogated with single particle tracking of quantum dots and FRAP in the context of neuronal cultures, where dendrite morphology and molecular composition have been radically altered from in vivo conditions. The rise of new optogenetic techniques, which provide great spatial and temporal precision, affords an opportunity to study mobility in more natural and intact conditions such as the acute slice preparation. Here we extend the optogenetic strategy to GABAARs, creating a light-regulated GABAAR (LiGABAR) by conjugating a photo-switchable tethered ligand (PTL) onto a mutant receptor containing a cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500-nm and 380-nm light, respectively, resulting in photo-switchable receptor antagonism. We created a transgenic knock-in α1 photoswitch-ready mouse (α1 PhoRM) to express the mutant receptor at endogenous levels and distributions.

We make use of LiGABAR technology to pioneer a novel electrophysiological technique for measuring GABAAR mobility, which we call CREAP. We use this method to functionally probe GABAAR mobility in the context of acute brain slices and find that α1-containing receptors are a highly mobile population GABAARs that constitute a fluid portion of inhibitory synapses.