Target-dependent feedforward inhibition mediated by short-term synaptic plasticity in the cerebellum

Abstract

Cerebellar feedforward inhibition (FFI) is mediated by two distinct pathways targeting different subcellular compartments of Purkinje cells (PCs). The axon of the granule cell, the parallel fiber, makes excitatory synapses not only onto PCs but also onto two types of interneurons, basket and stellate cells. Basket and stellate cells then send inhibitory signals to the soma and dendrites of Purkinje cells, respectively. Functional differences between somatic and dendritic FFI pathways, however, remain unknown. Here we address this question by examining how basket and stellate cells are recruited dynamically under high-frequency granule cell inputs at mice cerebellum. Short-term plasticity of various synapses within the FFI circuit has been explored. Unexpectedly, the parallel fiber synapse, which was considered to be facilitating during repetitive stimulation, shows depression, when the postsynaptic target is a basket cell. Other factors in the FFI circuit, such as firing properties of interneurons and dynamics of inhibitory synapses, are similar between somatic and dendritic pathways. The target-dependent parallel fiber synaptic plasticity has functional consequences for the two FFI pathways, because we observe that PCs receive transient somatic inhibition during 50 Hz stimulation of granule cells but persistent dendritic inhibition.

Figure 1.

FFI circuits activated by granule cell stimulation. A, An illustration of somatic and dendritic FFI by PF → BC → PC pathway and PF → SC → PC pathway. B, A diagram showing granule cell extracellular stimulation and somatic patch-clamp recording. C, A single extracellular stimulation applied to the granule cell layer elicited a PSC recorded from the Purkinje cell soma. An excitatory current (negative) is followed by an inhibitory current (positive) in the control condition (black). The GABA_A receptor antagonist SR 95531 blocks the inhibitory current (gray). Recorded cells were voltage clamped at −55 mV. Each trace is an average of 10 sweeps, and stimulus artifacts are removed from PSC traces. D, Trains of stimulation pulses were applied to the granule cell layer. An example of a 50 Hz train and its responses are shown in a similar way to C. B, Basket cell; P, Purkinje cell; S, Stellate cell.

Figure 2.

Target-dependent plasticity of granule cell synapses. A, Imaging of simultaneous patch-clamp recording from Purkinje cell and basket cell (scale bar, 10 μm). EPSCs recorded from the BC during 50 Hz GC stimulations are depressing, and those recorded from the PC are facilitating. Cells were voltage clamped at −65 mV and in the presence of SR 95531. Each trace is an average of 10 sweeps. B, EPSCs of an SC under the same experiment condition as in A. C, Peak amplitudes of EPSCs recorded during the train are normalized to the first one and plotted against the number of responses. Each trace represents an average from 14 PCs (open squares), 13 BCs (filled circles), and 13 SCs (open circles) (mean ± SEM).

Figure 3.

Deletion of Munc13-3 enhances the paired-pulse facilitation at GC → BC synapse, but GC → PC and GC → SC synapses are unaltered. A, An example trace of EPSCs recorded from a basket cell of Munc13-3 KO mice (top) shows no depression. The cell was voltage clamped at −65 mV in the presence of SR 95531, and 50 Hz trains of stimulation were applied to the granule cell layer. Normalized EPSCs were averaged from six recordings in KO mice (open circles in bottom) present a significant increase of the paired-pulse ratio compared with their wild-type littermates (filled circles). *p < 0.01. B, The same as A, but the postsynaptic cells were Purkinje cells and stellate cells.

Figure 4.

Spiking of interneurons in FFI circuit. A, A 200 pA current injection to the soma of BCs and SCs induced 140 Hz regular spike output of the example BCs and 165 Hz regular spike output of the SCs (top). Averaged spike counts within 500 ms were plotted against the amplitudes of injected currents (bottom). B, Spikes were detected from BCs and SCs and plotted as a raster plot during a 50 Hz train of granule cell stimulation. Spikes were counted from a 5 ms time window after each stimulation pulse. Spiking probabilities are plotted beneath the raster plot. Summarized spiking probability is shown for both cell types (bottom).

Figure 5.

Depressing inhibitory synapses in FFI circuit. A, A 50 Hz current pulse injection to a BC or SC elicited a reliable spike during each pulse (spikes were not shown). Simultaneous voltage-clamp recording with 135 mm intracellular [Cl⁻] from a PC that was innervated by the interneuron shows inward inhibitory current (negative) with depressing dynamics. Holding potential was −70 mV. B, Normalized IPSCs are plotted against the number of responses showing similar dynamics of these two synapses (mean ± SEM).

Figure 6.

SC → PC connection with realistic firing pattern of SC under 50 Hz GC stimulation. A, Firing patterns of an example SC in response to the GC stimulation at 50 Hz (Fig. 4B) was applied to stellate cells. Twenty different patterns were applied, and the resulting IPSCs from the PC were averaged (bottom trace). B, The summary of normalized peak amplitudes from seven cells.

Figure 7.

Persistent dendritic inhibitory potentials. A, PSPs were recorded from the dendritic patch pipette during a 50 Hz granule cell stimulation in both control (black) and in the presence of SR 95531 (gray) conditions (top). Each PSP was offset by the decay of its previous response to highlight the difference between the two conditions during each stimulation (bottom). B, Subtracting the two traces resulted in IPSPs that were blocked by SR 95531 (top). The peak value of each IPSP during the train was normalized to the first one. An average of three cells is plotted against the number of responses showing 80 ± 4.7% steady-state depression. Resting membrane potential at the dendrite was −60.5 ± 1.5 mV (control) and −64.2 ± 2.1 mV (SR 95531). Resting membrane potential at the soma was −59.5 ± 0.7 mV (control) and −62.3 ± 2.4 mV (SR 95531).

Figure 8.

Phasic somatic inhibition. A, Somatic recorded PSCs from a PC under 50 Hz GC stimulations are shown in both control (black) and SR 95531 (blue) conditions (top). Subtracting the two traces gave an estimation of somatic IPSCs (red). Normalized IPSCs were averaged from six cells showing 48 ± 1.5% steady-state depression. B, The same experiment as in A, but the data were obtained from Munc13-3 KO mice.

Figure 9.

Silencing a single basket cell indicates phasic somatic inhibition. A diagram showing the experimental scheme of silencing one BC by voltage clamping the BC at −70 mV during a paired recording of BC and PC (left). Somatic PSCs recorded from the PC under 50 Hz GC stimulations are shown in control condition (black, when the BC is under current-clamp mode) and in the condition when the BC is silenced (gray). Subtracting the two traces shows the IPSCs delivered by one BC onto the soma of the PC (bottom panel, right). Zero level is indicated as a dotted line. An average of 40 ± 5.8% steady-state depression was observed from four cells. B, Basket cell; P, Purkinje cell; S, Stellate cell.