Fast Times: Excitatory Effect of GABA in Axonal Compartments in the Cerebellar Molecular Layer

While exploring the functions of individual cells in a circuit, we can often overlook the importance of specialized subcellular compartments organized within one neuron. We probe the former with techniques like optogenetics, optical imaging, and (multi) cell recordings, while the latter can be clouded with mystery that have only begun to resolve in recent years. We explore these seemingly remote and difficult-to-record-from places in a well-mapped circuit in the cerebellum that is also relevant to behavior. Granule cells of the cerebellar cortex carry information associated with the context of a motor movement to the Purkinje cells, which as the sole output of the cerebellar cortex, is a major site of motor learning. Traditionally thought to be inhibitory, GABAA receptors (GABAARs) in granule cells, although inhibitory in the soma, have been found to be excitatory in the axons. These axons, the parallel fibers, are too thin to record from, and methods to study them include population recordings like calcium imaging and fiber volley detection, computational modeling, and voltage sensitive dyes.We first studied the presynaptic terminals of parallel fibers using calcium imaging, and showed that GABAAR activation leads to increased amplitude of stimulus-evoked calcium transients. This excitation appears to bring axons closer to firing threshold, recruiting addition fibers as opposed to increasing calcium levels on a per fiber basis. Blocking the transporter that accumulates chloride reduces the effect, suggesting that high chloride concentration in the axons is the mechanism behind this effect.To gain better temporal resolution, we used GABA uncaging to probe extracellular fiber volleys and found that GABA not only increases fiber volley amplitudes, but also increases conduction velocity on parallel fibers. We showed that δ-subunits are not required for GABAAR-mediated excitation and that endogenous GABA in a slice is sufficient to excite parallel fibers. Using a computational model of the granule cell, we showed how GABA-mediated excitation on the axons can influence cell spiking, and that properties of sodium channel inactivation determine whether there's an excitatory effect of GABA on threshold for spiking and on conduction velocity.Finally we described a novel method for detecting voltage in subcellular compartments utilizing fluorescence transfer between a lipophilic tracer dye and a voltage sensitive compound. This two-component system is seen to produce some of the biggest voltage sensing signals in the literature, and is capable of recording submillisecond voltage fluctuations in subcompartments of a Purkinje cell. We propose to use this system to record voltage from parallel fibers and determine the effect of GABA on action potential shape and subthreshold voltage fluctuations