Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition Ian Duguid1,2,3, Tiago Branco1,4, Paul Chadderton5, Charlotte Arlt, Kate Powell6, and Michael Häusser3 Wolfson Institute for Biomedical Research and Department of Neuroscience, Physiology, and Pharmacology, University College London, London WC1E 6BT, United Kingdom Edited by Masao Ito, RIKEN Brain Science Institute, Wako, Japan, and approved September 1, 2015 (received for review May 25, 2015) Classical feed-forward inhibition involves an excitation–inhibition Results sequence that enhances the temporal precision of neuronal re- Sensory-Evoked Phasic and Spillover Golgi Cell Inhibition Precedes sponses by narrowing the window for synaptic integration. In Mossy Fiber Excitation in Granule Cells. Cerebellar granule cells the input layer of the cerebellum, feed-forward inhibition is thought receive direct phasic and indirect or “spillover” GABAergic in- to preserve the temporal fidelity of granule cell spikes during mossy put from Golgi cells (6, 16, 20, 21). To investigate the temporal fiber stimulation. Although this classical feed-forward inhibitory cir- dynamics of sensory-evoked inhibition in vivo, we recorded cuit has been demonstrated in vitro, the extent to which inhibition spontaneous and sensory-evoked excitatory (Vhold = −70 mV) shapes granule cell sensory responses in vivo remains unresolved. and inhibitory (Vhold = 0 mV) currents from the same granule Here we combined whole-cell patch-clamp recordings in vivo and cells in Crus II (Fig. 1 A–D). Granule cells were identified based dynamic clamp recordings in vitro to directly assess the impact of on their characteristic electrophysiological properties (Table S1), Golgi cell inhibition on sensory information transmission in the depth from the pial surface (>250 μm), and morphology (Fig. granule cell layer of the cerebellum. We show that the majority 1B). To evoke behaviorally relevant somatosensory input, we of granule cells in Crus II of the cerebrocerebellum receive sensory- applied brief air puffs (60 ms) to the whiskers and ipsilateral evoked phasic and spillover inhibition prior to mossy fiber excita- perioral surface (22, 23). Sensory stimulation triggered bursts of tion. This preceding inhibition reduces granule cell excitability and mossy fiber excitatory postsynaptic currents (EPSCs; 4.9 ± 0.6 sensory-evoked spike precision, but enhances sensory response events; burst frequency, 104.0 ± 10.3 Hz; burst duration, 54.8 ± 4.0 reproducibility across the granule cell population. Our findings ms) (23–25). The same sensory stimulus also triggered phasic in- NEUROSCIENCE suggest that neighboring granule cells and Golgi cells can receive hibitory postsynaptic currents (IPSCs; 5.6 ± 0.6 events; burst segregated and functionally distinct mossy fiber inputs, enabling Golgi frequency, 65.5 ± 8.8 Hz; burst duration, 93.3 ± 15.1 ms) in the cells to regulate the size and reproducibility of sensory responses. same granule cells (n = 9 of 9 cells) (Fig. 1 C and D). Each cerebellum | Golgi cells | granule cells | inhibition | synaptic integration Significance lassical feed-forward inhibition (FFI) involves a sequence of Understanding how synaptic inhibition regulates sensory re- Cexcitation rapidly terminated by inhibition. This temporal sponses is a fundamental question in neuroscience. In cere- sequence narrows the time window for synaptic integration and bellar granule cells, sensory stimulation is thought to evoke an enforces precise spike timing (1–7). FFI is thought to be im- excitation–inhibition sequence driven by direct input from portant for regulating the temporal fidelity of spike responses in mossy fibers and followed by classical disynaptic feed-forward many neural systems, including the motor system, where rapid inhibition from nearby Golgi cells. We made, to our knowl- and adaptable changes in muscle activity are essential for coor- edge, the first voltage-clamp recordings of sensory-evoked dinated motor control (8–10). The cerebellum plays a central inhibition in granule cells in vivo and show that, surprisingly, role in fine sculpting of movements, and damage to the cerebellum sensory-evoked inhibition often precedes mossy fiber excita- produces severe motor deficits, most notably enhanced temporal tion activated by the same stimulus. We demonstrate how such “ ” variability of voluntary movements (11, 12). These findings suggest preceding inhibition can shape granule cell responses to that cerebellar circuits have the ability to preserve precise timing sensory stimulation. Our findings challenge the existing view that classical feed-forward inhibition is the dominant mode of information during behavior (5, 6, 13), and in vitro studies have inhibition, suggesting that parallel inhibitory networks regu- shown that feed-forward inhibitory networks in the input layer of late sensory information transmission through the granular layer. the cerebellum provide a mechanism for maintaining the temporal fidelity of information transmission (6, 14, 15). Author contributions: I.D., T.B., P.C., C.A., K.P., and M.H. designed research; I.D., T.B., P.C., Synaptic inhibition in the granule cell layer is generated by C.A., and K.P. performed research; I.D. and T.B. analyzed data; and I.D., T.B., and M.H. Golgi cells, GABAergic interneurons that provide direct in- wrote the paper. hibitory input to granule cells (6, 15–17). The prevailing view is The authors declare no conflict of interest. that, when mossy fibers are activated, granule cells receive both This article is a PNAS Direct Submission. monosynaptic excitation and disynaptic FFI from Golgi cells, Freely available online through the PNAS open access option. providing temporally precise inhibitory input that narrows the 1I.D. and T.B. contributed equally to this work. window for the temporal summation of discrete mossy fiber 2 – Present address: Centre for Integrative Physiology, School of Biomedical Sciences, Uni- inputs (6, 14, 18). This classical excitation inhibition sequence versity of Edinburgh, Edinburgh EH8 9XD, Scotland. forms the basis of a variety of contemporary cerebellar models 3To whom correspondence may be addressed. Email: [email protected] or m.hausser@ (7, 9, 18, 19). However, the exact temporal relationship be- ucl.ac.uk. tween sensory-evoked excitation and inhibition in granule cells 4Present address: Medical Research Council Laboratory of Molecular Biology, Cambridge has never been determined in vivo. Here, we combined in vivo CB2 0QH, United Kingdom. whole-cell voltage-clamp recordings from granule cells and 5Present address: Department of Bioengineering, South Kensington Campus, Imperial in vitro dynamic clamp experiments to investigate both the College London, London SW7 2AZ, United Kingdom. temporal dynamics of Golgi-cell–mediated inhibition and its 6Present address: UCL Institute of Ophthalmology, London EC1V 9EL, United Kingdom. importance for shaping sensory responses in the input layer of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the cerebellum. 1073/pnas.1510249112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1510249112 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 GoC the excitatory synaptic conductance measured in the same granule A PF B cell (Fig. S1). After the short latency excitatory/inhibitory responses in granule cells, we observed a prolonged reduction in spontaneous IPSC rate (duration, 340.0 ± 89.9 ms, n = 6/9 cells), reflecting long- GC lasting pauses in Golgi cell firing after sensory stimulation (28, 29). Air puff Given that Golgi cells fire one or two temporally precise spikes MF1 during the onset of sensory stimulation (28, 29)—albeit with vari- Stimulus Stimulus able onset latencies (28, 29)—and that each granule cell receives C (-70 mV) D 50 ms direct input from at least five to seven Golgi cells (21, 30), our 15 pA (0 mV) data are consistent with sensory-evoked inhibition being the re- 15 pA sult of pooled input from multiple Golgi cells. 50 ms To investigate whether evoked IPSCs occurred according to the classical excitation–inhibition sequence (6), we examined the relative timing of EPSCs and IPSCs in the same cell during sensory stimulation. Surprisingly, in the majority of granule cells, the mean onset latency of sensory-evoked inhibition was shorter 250 ms 250 ms than the latency of direct mossy fiber input evoked by the same 1.6 ± ± 3.0 sensory stimulus (IPSC latency, 10.5 1.1 ms; EPSC latency, 14.6 1.2 2.2 ms; n = 9), contrary to the expectation for a strictly feed-forward 2.0 0.8 pathway (Fig. 1 F–I). This result suggests that sensory-evoked phasic 1.0 0.4 IPSCs per bin EPSCs per bin inhibition of granule cells is mediated by Golgi cells activated by a 0.0 0.0 0 500 1000 1500 2000 0 500 1000 1500 2000 subset of mossy fibers distinct from those providing direct mono- Time (ms) Time (ms) synaptic granule cell excitation (i.e., FFI) or disynaptic feed-forward Sensory-evoked Phasic Spillover excitation from granule cells (i.e., feedback inhibition) (15, 31–34). E (spillover subtracted) (phasic subtracted) Importantly, this reversed temporal relationship did not depend on the anesthetic regime used (Fig. S2). Given that brief stimulation of the upper lip and perioral surface has been shown to generate precise, short-latency (∼7–10 ms) output from Golgi cells (28, 29) Stimulus Stimulus Stimulus 10 pA ∼ – 30 ms and highly variable, longer-latency ( 15 25 ms) excitatory input in granule cells (Fig. 1F) (23), our data suggest that some Golgi cells Stimulus Stimulus FIG H 30 -10 receive direct trigeminal input before neighboring granule cells re- )sm ) s 25 ( ycn ( m ceiving delayed corticopontine input (35, 36). ( ycne 20 -5 0 mV etal t -70 mV al 15 tesno ts tesno CSPI CSPI Properties of Golgi Cell Inhibition During Sustained Sensory-Evoked 10 0 - - Mossy Fiber Input. Mossy fiber input to the granule cell layer can C ruB 5 S PE occur in short high-frequency bursts (24, 29, 37) or as sustained, 10 pA 10 pA 0 5 – 20 ms 10 ms IPSC EPSC time-varying synaptic input (22, 38 40), depending on the nature of the stimulus.