Research Articles: Cellular/Molecular Calcium channel-dependent induction of long- term synaptic plasticity at excitatory Golgi cell synapses of cerebellum https://doi.org/10.1523/JNEUROSCI.3013-19.2020 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.3013-19.2020 Received: 20 December 2019 Revised: 15 December 2020 Accepted: 18 December 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 the authors 1 Calcium channel-dependent induction of long-term synaptic plasticity 2 at excitatory Golgi cell synapses of cerebellum 3 4 F. Locatelli1*, T. Soda1,3*, I. Montagna1, S. Tritto1, L. Botta4, F. Prestori1, E. D'Angelo1,2 5 6 1Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy 7 2Brain Connectivity Center, IRCCS Mondino Foundation, Pavia, Italy 8 3Museo Storico della Fisica e Centro Studi e Ricerche Enrico Fermi, Rome, Italy 9 4Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Pavia, Italy 10 * FL and TS are co-first authors 11 12 Correspondence should be addressed to: 13 Prof. Egidio D'Angelo 14 [email protected] 15 and 16 Dr. Francesca Prestori 17 [email protected] 18 Dept of Brain and Behavioral Sciences 19 via Forlanini 6, 27100 Pavia, Italy 20 [email protected] 21 22 Number of pages: 25 23 Number of tables: 1 24 Number of figures: 7 25 Number of words: 209-Abstract, 546-Introduction, 1325-Discussion 26 27 Running Title: Golgi cell bidirectional plasticity 28 Key words: cerebellum, Golgi cell, synaptic plasticity, Ca2+ channels 29 1 30 Acknowledgments. 31 This project/research received funding from the European Union’s Horizon 2020 Framework 32 Programme for Research and Innovation under the Framework Partnership Agreement No. 650003 33 (HBP FPA) to ED. This research was supported by the HBP Brain Simulation Platform, funded 34 from the European Union’s Horizon 2020 Framework Programme for Research and Innovation 35 under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2) and by the 36 EBRAINS research infrastructure, funded from the European Union’s Horizon 2020 Framework 37 Programme for Research and Innovation under the Specific Grant Agreement No. 945539 (Human 38 Brain Project SGA3) to ED. This research was also supported by the MNL Project “Local Neuronal 39 Microcircuits” of the Centro Fermi (Rome, Italy) to ED 40 Special thanks to Javier DeFelipe (Department of Neuroanatomy and Cell Biology, Instituto Cajal 41 (CSIC), Madrid, Spain) for Golgi cell morphological reconstruction. 42 43 Author contribution. 44 FL and TS performed research; FL, TS, IM and FP analyzed data; ST performed microscopy cell 45 reconstruction; LB performed mice genotyping; FP and ED wrote the paper; ED designed research. 46 47 Abstract 48 The Golgi cells, together with granule cells and mossy fibers, form a neuronal microcircuit 49 regulating information transfer at the cerebellum input stage. Despite theoretical predictions, little 50 was known about long-term synaptic plasticity at Golgi cell synapses. Here we have used whole- 51 cell patch-clamp recordings and calcium imaging to investigate long-term synaptic plasticity at 52 excitatory synapses impinging on Golgi cells. In acute mouse cerebellar slices, mossy fiber theta- 53 burst stimulation (TBS) could induce either long-term potentiation (LTP) or long-term depression 54 (LTD) at mossy fiber-Golgi cell and granule cell-Golgi cell synapses. This synaptic plasticity 55 showed a peculiar voltage-dependence, with LTD or LTP being favored when TBS induction 56 occurred at depolarized or hyperpolarized potentials, respectively. LTP required, in addition to 57 NMDA channels, activation of T-type Ca2+ channels, while LTD required uniquely activation of L- 58 type Ca2+ channels. Notably, the voltage-dependence of plasticity at the mossy fiber-Golgi cell 2 59 synapses was inverted with respect to pure NMDA receptor-dependent plasticity at the neighboring 60 mossy fiber-granule cell synapse, implying that the mossy fiber presynaptic terminal can activate 61 different induction mechanisms depending on the target cell. In aggregate, this result shows that 62 Golgi cells show cell-specific forms of long-term plasticity at their excitatory synapses, that could 63 play a crucial role in sculpting the response patterns of the cerebellar granular layer. 64 65 Significance statement 66 This paper shows for the first time a novel form of Ca2+ channel-dependent synaptic 67 plasticity at the excitatory synapses impinging on cerebellar Golgi cells. This plasticity is 68 bidirectional and inverted with respect to NMDA receptor-dependent paradigms, with LTD and 69 LTP being favored at depolarized and hyperpolarized potentials, respectively. Furthermore, LTP 70 and LTD induction requires differential involvement of T-type and L-type voltage-gated Ca2+ 71 channels rather than the NMDA receptors alone. These results, along with recent computational 72 predictions, support the idea that Golgi cell plasticity could play a crucial role in controlling 73 information flow through the granular layer along with cerebellar learning and memory. 74 75 Introduction 76 Several forms of plasticity are thought to provide the substrate for learning and memory in 77 brain microcircuits (Bliss et al., 2014; Volianskis et al., 2015). In the cerebellum, which is involved 78 in motor learning and cognitive processing (Marr, 1969; Ito, 2008; Koziol et al., 2014; Sokolov et 79 al., 2017; D'Angelo, 2019), more than 15 diverse forms of long-term synaptic and non-synaptic 80 plasticity have been reported at several sites across the granular layer, molecular layer and deep 81 cerebellar nuclei (Hansel et al., 2001; Gao et al., 2012; D'Angelo et al., 2016). In the granular layer, 82 non-synaptic plasticity has been shown both in granule cells (Armano et al., 2000) and Golgi cells 83 (Hull et al., 2013), while long-term synaptic plasticity has been reported at the synapses made by 84 mossy fibers with granule cells (Armano et al., 2000; Medina and Mauk, 2000; Zhang and Linden, 85 2006; D'Errico et al., 2009; Pugh and Raman, 2009; D'Angelo, 2014; Sgritta et al., 2017; Moscato 86 et al., 2019). However, long-term synaptic plasticity elicited by mossy fiber stimulation in Golgi 87 cells has not been investigated yet. 88 Golgi cells are the main inhibitory interneurons of the granular layer, where they receive 89 excitatory inputs from mossy fibers and granule cells (Palay, 1974). The mossy fibers make 90 synapses on Golgi cell basolateral dendrites inside the cerebellar glomeruli, which also contact the 91 dendrites of granule cells. The granule cells, in turn, make synapses both on the Golgi cell 92 basolateral dendrites through their ascending axons and on the apical dendrites through the parallel 3 93 fibers. All these excitatory synapses are glutamatergic and express AMPA and NMDA receptors 94 activated during synaptic transmission (Misra et al., 2000; Cesana et al., 2013). Golgi cells also 95 receive inhibitory innervation from neighboring Golgi cells and other inhibitory interneurons 96 (Dieudonne, 1998; Bureau et al., 2000; Misra et al., 2000; Hull and Regehr, 2012). Finally, Golgi 97 cell axons contact granule cell dendrites inside the glomeruli, thus regulating information flow to 98 the cerebellar cortex trough a mix of feedback and feed forward inhibition (D'Angelo, 2008; 99 Kanichay and Silver, 2008; Cesana et al., 2013; D'Angelo et al., 2013). It has been suggested that 100 Golgi cells dynamically control the gain and the temporal pattern of granule cell discharge in 101 response to mossy fiber activity (Marr, 1969; Mitchell and Silver, 2003; D'Angelo and De Zeeuw, 102 2009; Billings et al., 2014) and this essential role has been supported by their acute ablation, which 103 causes severe motor deficits and ataxia (Watanabe et al., 1998). 104 Golgi cells are low-frequency pacemakers (Dieudonne, 1998; Forti et al., 2006) and they 105 have recently been shown to express Ca2+ channels in the dendrites (Rudolph et al., 2015). The Ca2+ 106 channels, along with postsynaptic NMDA receptors, may actually enable the induction of long-term 107 synaptic plasticity, as observed at other synapses (Lisman, 1989; Shouval et al., 2002; Volianskis et 108 al., 2015; Leresche and Lambert, 2017). In this work we have combined patch-clamp recordings 109 and calcium imaging techniques to show, for the first time, the existence of bidirectional long-term 110 plasticity at the excitatory Golgi cell synapses activated by patterned mossy fiber stimulation. These 111 results support recent computational predictions about the critical role that such forms of plasticity 112 would play in controlling learning and computation in the cerebellar granular layer (Schweighofer 113 et al., 2001; Garrido et al., 2013; Garrido et al., 2016). 114 115 Methods 116 The experiments have been performed on 16-to 21-day-old (P0=day of birth) GlyT2-GFP 117 mice (of either sex) heterozygous for the bacterial artificial chromosome insertion of EGFP under 118 the glycine transporter type 2 gene (Zeilhofer et al., 2005). All procedures were conducted in 119 accordance with European guidelines for the care and use of laboratory animals (Council Directive 120 2010/63/EU), and approved by the ethical committee of Italian Ministry of Health (628/2017-PR). 121 The mice were anesthetized with halothane (Sigma-Aldrich) and killed by decapitation in order to 122 remove the cerebellum for acute slice preparation according to established techniques (Forti et al., 123 2006; Cesana et al., 2013). 124 125 126 4 127 Slices preparation and solutions 128 The cerebellar vermis was isolated and fixed on the vibroslicer’s stage (Leica VT1200S; 129 Leica Biosystems) with cyano-acrylic glue.