Two-photon calcium imaging reveals long-term changes in cerebellar granule cell responsiveness following high-frequency mossy fibers stimulation
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1
University of Pavia, Dept. of Brain and Behavioral Sciences, Italy
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2
Brain Connectivity Center, C. Mondino National Neurological Institute, Italy
Recent years have witnessed major improvements in our understanding of the brain, thanks to technological developments and a new multidisciplinary approach. Single-cell studies have allowed researchers to gain insights into the detailed characteristics of different neuronal types, while techniques such as functional magnetic resonance imaging (fMRI) enabled an investigation of the global activity of the brain. However, very little is known about the microcircuits function and the way in which each single cell contributes to the final output. In order to investigate the spatio-temporal organization of neuronal activity in local microcircuits, a simultaneous and fast recording from selected multiple neurons with a single-cell resolution is required. Optical techniques fit this purpose, but a parallel and fast signals detection requires the presence of multiple confocal excitation volumes that cannot be achieved through traditional confocal and two-photon microscopy.
The use of a Spatial Light Modulator (SLM) to divide a coherent excitation light in multiple diffraction limited focal points which can be configured in different patterns enabled us to perform optical recordings from different neurons simultaneously. We recently developed an SLM-two photon microscope (SLM-2PM) able to resolve the spatio-temporal organization of activity in acute cerebellar slices through the simultaneous acquisition of calcium signals from multiple granule cells (GrCs, Fig. 1A, B, C). The synaptic origin of calcium signals was assessed as due to glutamate receptors activation and a quasi-linear correlation between the number of GrCs action potentials (APs) and the fluorescence signals was established through patch-clamp whole-cell recordings (WCRs) (Fig. 1D) [1, 2]. However, it was unclear whether SLM-2PM could be used to investigate long-term synaptic plasticity.
In this work, we optimized the SLM-2PM to investigate the effect of a mossy fibers (mfs) high-frequency stimulation protocol (100 pulses at 100 Hz, HFS), that is known to induce long-term potentiation and depression (LTP and LTD) in the cerebellar granular layer [3, 4, 5].
Parasagittal acute cerebellar slices (220 μm) were obtained from 19 to 24 days old Wistar rats. For bulk loading of slices a 50 μg aliquot of Fura-2 AM (previously dissolved in 48 CL of Dimethyl Sulfoxied, DMSO, and in 2 μL of Pluronic F-127) was mixed with 2 mL of continuously oxygenated Krebs solution. Slices were maintained in this solution at 35°C for 30 minutes in the dark and then positioned in the recording chamber of the microscope. Oxygenated Krebs solution (2 mL/min) maintained at 30°C and added with a GABA-A receptors blocker (10 μM gabazine, SR95531, Abcam) was perfused during the whole experiments. GrCs activity was elicited by mfs stimulation through a large-tip (10-20 μm) patch-pipette filled with extracellular Krebs solution, via a stimulus isolation unit. The same stimulation protocol (10 pulses at 50 Hz) was applied before and after the delivery of the HFS protocol. The number of GrCs that showed long-term plasticity was evaluated by considering persistent fluorescence variations of ± 20% after the induction.
We observed long-term changes of GrCs calcium responses after the HFS protocol both as potentiation and as depression (CaR-P and CaR-D), as well as GrCs showing no changes in signals amplitude. CaR-P and CaR-D showed impressive variations in signal amplitude (+368.7 ± 33.9%, n=12, -73.1 ± 3.6%, n=4, respectively, 30 minutes after HFS, Fig. 2). Interestingly, CaR-P amplitude changes were several times larger than those reported for synaptic currents measurements [6, 7].
To elucidate the underlying mechanism we performed preliminary patch-clamp experiments in loose cell-attached (LCA) configuration (10-50 MΩ seal resistance). Stimulus-induced GrC APs were evoked through the same stimulation protocol used in SLM-2PM experiments, in order to elicit the same cellular behavior (10 pulses at 50 Hz, before and after the plasticity induction). Neuronal activity was analyzed off-line as number of stimulus-induced APs; long-term plasticity was evaluated by considering statistically significant changes in the average number of APs after the induction. GrCs activity showed prolonged changes comparable to those observed with SLM-2PM calcium imaging recordings (+105.5 ± 6.7%, n=7, -73.8 ± 3.0%, n=6). In order to visualize the different trends simultaneously, in Fig. 3A the corresponding normalized time course is shown. The LTP magnitude reached in LCA experiments showed a dependency on the pre-stimulation state similar to that observed in previous results obtained in our lab [7]. Through WCRs, indeed, an indirect relationship between the initial probability (p) of quantum release and the LTP magnitude shown after plasticity induction was established (Fig. 3B). Preliminary LCA recordings reveal a similar correlation between the probability of APs generation in response to a single mf stimulus (positively correlated to a high p value) and the LTP magnitude (R^2 = 0.7, n = 7, Fig. 3C).
Long-term plasticity SLM-2PM experiments revealed persistent changes of calcium signals amplitudes, both as potentiation and as depression (CaR-P and CaR-D). The amplitude changes observed in CaR-P could be explained by an increase in the probability of APs generation after LTP induction [3], suggesting that CaR-P and CaR-D most likely reflect a combination of changes in GrCs intrinsic excitability and in synaptic transmission. Ongoing LCA experiments are helping to elucidate the underlying mechanisms, allowing to provide further insight into the understanding of neuronal plasticity.
Acknowledgements
This work was supported by: European Union grant Human Brain Project (HBP-29 604102) to ED. The authors declare no competing financial interests.
References
[1] Gandolfi D., Pozzi P., Tognolina M., Chirico G., Mapelli J. and D'Angelo E. (2014) “The spatiotemporal organization of cerebellar network activity resolved by two-photon imaging of multiple single neurons”. Frontiers in cellular neuroscience 8(92);
[2] Pozzi P., Gandolfi D., Tognolina M., Chirico G., Mapelli J. and D'Angelo E. (2015) “High-throughput spatial-light-modulation two-photon microscopy for fast functional imaging” . Neurophotonics 2:1;
[3] Armano S., Rossi P., Taglietti V., D’Angelo E. (2000) “Long-term potentiation of intrinsic excitability at the mossy fiber – granule cell synapse of rat cerebellum. The Journal of Neuroscience”, 20(14);
[4] Gall D., Prestori F., Sola E., D’Errico A., Roussel C., Forti L., Rossi P., D’Angelo E. (2005) “Intracellular Calcium Regulation by Burst Discharge Determines Bidirectional Long-Term Synaptic Plasticity at the Cerebellum Input Stage”. The Journal of Neuroscience, 25(19);
[5] D’Errico., Prestori F. and D’Angelo E. (2009) “Differential induction of bidirectional long-term changes in neurotransmitter release by frequency-coded patterns at the cerebellar inpu”t. J Physiology, 587;
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Keywords:
two-photon calcium imaging,
Cerebellum,
spatial light modulator,
Long-term plasticity,
patch-clamp recording
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:
Tognolina
M,
Mapelli
L and
D‘Angelo
E
(2019). Two-photon calcium imaging reveals long-term changes in cerebellar granule cell responsiveness following high-frequency mossy fibers stimulation.
Conference Abstract:
The Cerebellum inside out: cells, circuits and functions
.
doi: 10.3389/conf.fncel.2017.37.00009
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Received:
30 Nov 2016;
Published Online:
25 Jan 2019.
*
Correspondence:
Miss. Marialuisa Tognolina, University of Pavia, Dept. of Brain and Behavioral Sciences, Pavia, Italy, marialuisa.tognolina01@universitadipavia.it
Dr. Lisa Mapelli, University of Pavia, Dept. of Brain and Behavioral Sciences, Pavia, Italy, lisa.mapelli@unipv.it
Prof. Egidio D‘Angelo, University of Pavia, Dept. of Brain and Behavioral Sciences, Pavia, Italy, dangelo@unipv.it