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Anti-hebbian long-term synaptic plasticity at the mossy fiber- Golgi cell synapse of cerebellum

Francesca Locatelli1*, Teresa Soda1, 2, Francesca Prestori1* and Egidio D‘Angelo1, 3*
  • 1 University of Pavia, Dept.Brain and Behavioral Sciences, Italy
  • 2 Museo storico della Fisica e Centro Studi e Ricerche Enrico Fermi, Italy
  • 3 Brain Connectivity Center,C.Mondino National Neurological Institute, Italy

This study is focused on synaptic properties of Golgi cells (GoCs), the main inhibitory neurons of cerebellar granular layer, and the presence of long-term synaptic plasticity at the mossy fibers- Golgi cell (MFs-GoC) synapses was evaluated. GoCs are characterized by an irregular soma with a diameter of 20–40 μm from which radiate several basal dendrites, two to three apical dendrites and an extensively ramified axon [1]. The most relevant excitatory input to GoCs comes from the MFs arriving to the glomeruli, thus forming synapses on basal dendrites and allowing them to mediate feedforward inhibition (MFs→GoC→GrC). Moreover, the GoCs receives connection from GrCs principally through the PFs or, in alternative, through synapses en passant along their ascending axon, enabling a feedback inhibition (MFs→GrC→GoC→GrC). GoCs also receive inhibitory signals from molecular layer neurons [5-6]. Finally, recent studies revealed the existence of inhibitory GoC-GoC communication through gap-junctions [10]. Cerebellar inhibition results from feedback and feedforward loops shaping the temporal aspect and spatial organization of signals relayed to the molecular layer. Over the years, the cerebellum has been object of many investigations on different forms of synaptic plasticity and their mechanisms of induction, which grant a critical contribution to motor learning and have the function to regulate the overall level of activity in the cerebellar circuitry. In particular, studies revealed the existence of multiple forms of long-term plasticity in the molecular layer, granular layer and DCN, thus demonstrating that the plastic capability of the cerebellum is more complex and extended than initially expected.[2]. Therefore, considering the functional implications of GoCs for granular layer network, and the importance of plasticity at other synapses, it becomes crucial to evaluate the existence of forms of plasticity at the MF-GoC synapses. Recent mathematical models have implied a critical role for plasticity at GoC synapses but their existence remained unknown [9]. Here, this plasticity was investigated combinding patch clamp recordings and calcium imaging technique in acute cerebellar slices to clarify the GoCs role in the granular layer. Parasagittal cerebellar slices (220 µm thick) were cut from the vermis of 16- to 20 old transgenic mice that express enhanced green fluorescent protein under the control of the glycine transporter type 2 promoter (GlyT2-EGFP, [11]) in order to make GoCs easily recognizable. MFs bundle was stimulated using a large-bore patch pipette, filled with standard extracellular solution, at a test frequency of 0.1 Hz. After 10-min, eight bursts of 10 impulses at 100 Hz were repeated every 250 ms (TBS, [3]), in the current-clamp mode at different holding potentials (Vh; at-60 mV for LTP and -40 mV for LTD) and we have evaluated the changes induces by TBS in excitatory postsynaptic currents (EPSCs) after 30 min. During recordings the slice was maintained at 32°C. The stimulation pipette was positioned in the white matter at least 300 μm away from the soma of the recorded GoC, well above the extension of the apical GoC dentritic arbor, an appropriate position to evoke monosynaptic inputs exclusively from MFs and a disynaptic GrCs inputs, avoiding direct activation of GrCs axons [4]. Calcium imaging was performed as reported previously in this same preparation [8], by using Oregon green BAPTA-1 (OG1, Molecular Probes). Briefly, the intracellular solution was added with 0.2 mM OG1 in substitution to the EGTA/Ca2+ buffer . Ca 2+indicator dye was loaded in GoC at least for 5 min before starting all Ca2+ imaging recordings.. All stimulation protocols were separated by a minimum of 60sec to allow [Ca2+]i to return to basal level. For each experiment it was performed an off-line analysis of stimulus-induced fluorescence changes in the regions of interest (ROIs), drawn by eye in the first image of a sequence. Furthermore, background area of similar size close to cell was delimited to estimate background fluorescence. Surprisingly, in whole cell recordings, TBS train stimulation induced bidirectional long-term synaptic plasticity at MF – GoC synapse. TBS delivered at -41.0±4.0 mV caused long term depression (LTD; -36.0±9.0% change, n=4; p<0.05). TBS delivered at -56.8± 1.4 mV caused long-term potentiation (LTP; 49.4± 12.1% change, n=4; p<0.05). Fig 1A. Same experiments was performed with pharmacology blockers, to clarify the involvement of NMDA receptor and Ca2+ channels. Perfusion of either the NMDA receptor blocker, APV (50μM), or of the calcium channel blocker, MIBEFRADIL (10μM), abolished LTP but not LTD. Fig 1B-C. Calcium imaging recordings shows that, during LTP induction, the fluorescent signal related to increase in [Ca2+]i was high in the dendrites [(∆F/F0)max = 0.21 ± 0.04; (n=10)] compared to the one observed for LTD [∆F/F0max 0,11 ± 0.02; (n=10); P<0,001]-.These results are coherent with the fact that at synaptic level LTP is characterized by a significative increase in Ca2+ level. Fig.2A. Moreover, the amplitude of synaptic [Ca2+]i transients after induction was monitored in the presence of APV and MIBEFRADIL. MFs stimulation with APV perfusion determined the absence of LTP due to a lower Ca2+ transient [(∆F/F0)max = 0.03±0.006; n=11; P<0,001], whereas Ca2+ transient related to the appearance of LTD seemed not to be significantly affected, compared to the control [(∆F/F0)max = 0,085±0,03; n=11; P=0,15]-Fig.2B. Coherent with the perfusion of MIBEFRADIL in whole-cell recordings, MFs stimulation is unable to elicit a sufficient Ca2+ increase for the induction of LTP [(∆F/F0)max = 0.12±0.01, n=2; P=0,15]. The Ca2+ signal obtained by MF stimulation for the induction of LTD was (∆F/F0)max=0,10±0.01,(n=2;P=0,9), so that is interestingly similar to the value reported for LTP. Fig. 2C. In conclusion, this results demonstrates the existence of long-term synaptic plasticity between MFs and GoCs and shows that it depends on membrane potential during induction related to Ca2+ influx. Interestingly, the LTP/LTD rule is inverted respect to plasticity at MF-GrC relay. Pharmacology results suggests that LTP induction requires postsynaptic NMDA channels and LVA channels, which are de-inactivated at hyperpolarized potentials.

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Acknowledgements

This work was supported by: European Union grant Human Brain Project (HBP-29 604102) to ED and Fermi grant [13(14)] to ED and TS. The authors declare no competing financial interests.

References

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Keywords: golgi cells, Cerebellum, LTP and LTD, Patch-Clamp Techniques, Calcium influx

Conference: The Cerebellum inside out: cells, circuits and functions , ERICE (Trapani), Italy, 1 Dec - 5 Dec, 2016.

Presentation Type: poster

Topic: Cellular & Molecular Neuroscience

Citation: Locatelli F, Soda T, Prestori F and D‘Angelo E (2019). Anti-hebbian long-term synaptic plasticity at the mossy fiber- Golgi cell synapse of cerebellum. Conference Abstract: The Cerebellum inside out: cells, circuits and functions . doi: 10.3389/conf.fncel.2017.37.000027

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Received: 30 Nov 2016; Published Online: 25 Jan 2019.

* Correspondence:
Dr. Francesca Locatelli, University of Pavia, Dept.Brain and Behavioral Sciences, pavia, Italy, francesca.locatelli@unipv.it
Dr. Francesca Prestori, University of Pavia, Dept.Brain and Behavioral Sciences, pavia, Italy, francesca.prestori@unipv.it
Prof. Egidio D‘Angelo, University of Pavia, Dept.Brain and Behavioral Sciences, pavia, Italy, dangelo@unipv.it