Optogenetic Visualization of Presynaptic Tonic Inhibition of Cerebellar Parallel Fibers

Abstract

Tonic inhibition was imaged in cerebellar granule cells of transgenic mice expressing the optogenetic chloride indicator, Clomeleon. Blockade of GABA A receptors substantially reduced chloride concentration in granule cells due to block of tonic inhibition. This indicates that tonic inhibition is a significant contributor to the resting chloride concentration of these cells. Tonic inhibition was observed not only in granule cell bodies, but also in their axons, the parallel fibers (PFs). This presynaptic tonic inhibition could be observed in slices both at room and physiological temperatures, as well as in vivo, and has many of the same properties as tonic inhibition measured in granule cell bodies. GABA application revealed that PFs possess at least two types of GABA A receptor: one high-affinity receptor that is activated by ambient GABA and causes a chloride influx that mediates tonic inhibition, and a second with a low affinity for GABA that causes a chloride efflux that excites PFs. Presynaptic tonic inhibition regulates glutamate release from PFs because GABA A receptor blockade enhanced both the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs at the PF-Purkinje cell synapse. We conclude that tonic inhibition of PFs could play an important role in regulating information flow though cerebellar synaptic circuits. Such cross talk between phasic and tonic signaling could be a general mechanism for fine tuning of synaptic circuits.

Biophysical models reveal the relative importance of transporter proteins and impermeant anions in chloride homeostasis

“…Without active Clflux, which CCCs provide, there is no driving force for passive Cl -635 flux and hence no mechanism for [Cl -]i changes resulting from selective modification of a Cl -636 conductance. This is consistent with both classic (Misgeld et al, 1986;Thompson and Gähwiler, 1989) 637 and recent experimental findings (Berglund et al, 2016). 638 639 In our model, we find that elevating the activity of KCC2, the most active CCC in mature neurons (Ben-640 Ari, 2002), increases the driving force for Clby shifting the reversal potential for Clcloser to that of 641 K + .…”

Odor-Induced Multi-Level Inhibitory Maps inDrosophila

“…AC, antennal commissure; AN, antennal nerve; ALT, AL tract. tent with observations in mammalian neurons (Kuner and Augustine, 2000;Berglund et al, 2006) and might reflect the relatively slow rate of Clremoval from the neurons (Staley and Proctor, 1999;Berglund et al, 2009Berglund et al, , 2016. This slow recovery in the Clresponse might affect the excitability of the neuron for a period significantly outlasting the stimulation.…”

“…Therefore, it is difficult to conclude to what degree the dynamics of Cltransients quantitatively reflect the actual balance between Clinflux and intracellular Clremoval. However, a slow recovery of Clomeleon signals has also been observed in mammalian neurons (Kuner and Augustine, 2000;Berglund et al, 2006) and has been attributed to the slow removal of [Cl -] i by transporters rather than kinetic properties of the Clsensor (Staley and Proctor, 1999;Berglund et al, 2009Berglund et al, , 2016. It is therefore quite conceiv-able that the odor-evoked Cl-transients in OSNs indeed strongly outlast the actual stimulation.…”

“…Binding of Clto YFP reduces its absorbance, which results in a change of the YFP/CFP emission ratio proportional to [Cl -] i . The applicability of Clomeleon in vivo has been demonstrated in hippocampal slices (Berglund et al, 2006), retinal bipolar cells (Haverkamp et al, 2005;Duebel et al, 2006), thalamo-cortical neurons of mice (Glykys et al, 2009) and cerebellar granule cells (Berglund et al, 2016).…”

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

“…This study has important differences from our approach, since they could only measure bulk changes of [Cl − ] i concentration in a relatively large brain volume. This is also true for a more recent study by Berglund et al (49) on tonic GABAergic inhibition in the cerebellum, where the authors performed [Cl − ] i imaging at cellular level in slices and used bulk measurements in vivo. Finally, it is obvious that, in experiments with SuperClomeleon, it is not possible to make a correction for pH i differences and dynamic changes at the level of individual neurons even in vitro, which would be required for a valid estimate of [Cl − ] i based on the highly pH-sensitive Cl − signal of this indicator.…”