Computational models of neurotransmission at cerebellar synapses unveil the impact on network computation.

The neuroscientific field benefits from the conjoint evolution of experimental and computational techniques, allowing for the reconstruction and simulation of complex models of neurons and synapses. Chemical synapses are characterized by presynaptic vesicle cycling, neurotransmitter diffusion, and postsynaptic receptor activation, which eventually lead to postsynaptic currents and subsequent membrane potential changes. These mechanisms have been accurately modeled for different synapses and receptor types (AMPA, NMDA, and GABA) of the cerebellar cortical network, allowing simulation of their impact on computation. Of special relevance is short-term synaptic plasticity, which generates spatiotemporal filtering in local microcircuits and controls burst transmission and information flow through the network. Here, we present how data-driven computational models recapitulate the properties of neurotransmission at cerebellar synapses. The simulation of microcircuit models is starting to reveal how diverse synaptic mechanisms shape the spatiotemporal profiles of circuit activity and computation.

Discovering Microcircuit Secrets With Multi-Spot Imaging and Electrophysiological Recordings: The Example of Cerebellar Network Dynamics.

The cerebellar cortex microcircuit is characterized by a highly ordered neuronal architecture having a relatively simple and stereotyped connectivity pattern. For a long time, this structural simplicity has incorrectly led to the idea that anatomical considerations would be sufficient to understand the dynamics of the underlying circuitry. However, recent experimental evidence indicates that cerebellar operations are much more complex than solely predicted by anatomy, due to the crucial role played by neuronal and synaptic properties. To be able to explore neuronal and microcircuit dynamics, advanced imaging, electrophysiological techniques and computational models have been combined, allowing us to investigate neuronal ensembles activity and to connect microscale to mesoscale phenomena. Here, we review what is known about cerebellar network organization, neural dynamics and synaptic plasticity and point out what is still missing and would require experimental assessments. We consider the available experimental techniques that allow a comprehensive assessment of circuit dynamics, including voltage and calcium imaging and extracellular electrophysiological recordings with multi-electrode arrays (MEAs). These techniques are proving essential to investigate the spatiotemporal pattern of activity and plasticity in the cerebellar network, providing new clues on how circuit dynamics contribute to motor control and higher cognitive functions.

Cellular-resolution mapping uncovers spatial adaptive filtering at the rat cerebellum input stage.

Long-term synaptic plasticity is thought to provide the substrate for adaptive computation in brain circuits but very little is known about its spatiotemporal organization. Here, we combined multi-spot two-photon laser microscopy in rat cerebellar slices with realistic modeling to map the distribution of plasticity in multi-neuronal units of the cerebellar granular layer. The units, composed by ~300 neurons activated by ~50 mossy fiber glomeruli, showed long-term potentiation concentrated in the core and long-term depression in the periphery. This plasticity was effectively accounted for by an NMDA receptor and calcium-dependent induction rule and was regulated by the inhibitory Golgi cell loops. Long-term synaptic plasticity created effective spatial filters tuning the time-delay and gain of spike retransmission at the cerebellum input stage and provided a plausible basis for the spatiotemporal recoding of input spike patterns anticipated by the motor learning theory.

Parameter tuning differentiates granule cell subtypes enriching transmission properties at the cerebellum input stage.

The cerebellar granule cells (GrCs) are classically described as a homogeneous neuronal population discharging regularly without adaptation. We show that GrCs in fact generate diverse response patterns to current injection and synaptic activation, ranging from adaptation to acceleration of firing. Adaptation was predicted by parameter optimization in detailed computational models based on available knowledge on GrC ionic channels. The models also predicted that acceleration required additional mechanisms. We found that yet unrecognized TRPM4 currents specifically accounted for firing acceleration and that adapting GrCs outperformed accelerating GrCs in transmitting high-frequency mossy fiber (MF) bursts over a background discharge. This implied that GrC subtypes identified by their electroresponsiveness corresponded to specific neurotransmitter release probability values. Simulations showed that fine-tuning of pre- and post-synaptic parameters generated effective MF-GrC transmission channels, which could enrich the processing of input spike patterns and enhance spatio-temporal recoding at the cerebellar input stage.

High-throughput spatial light modulation two-photon microscopy for fast functional imaging.

The optical monitoring of multiple single neuron activities requires high-throughput parallel acquisition of signals at millisecond temporal resolution. To this aim, holographic two-photon microscopy (2PM) based on spatial light modulators (SLMs) has been developed in combination with standard laser scanning microscopes. This requires complex coordinate transformations for the generation of holographic patterns illuminating the points of interest. We present a simpler and fully digital setup (SLM-2PM) which collects three-dimensional two-photon images by only exploiting the SLM. This configuration leads to an accurate placement of laser beamlets over small focal volumes, eliminating mechanically moving parts and making the system stable over long acquisition times. Fluorescence signals are diffraction limited and are acquired through a pixelated detector, setting the actual limit to the acquisition rate. High-resolution structural images were acquired by raster-scanning the sample with a regular grid of excitation focal volumes. These images allowed the selection of the structures to be further investigated through an interactive operator-guided selection process. Functional signals were collected by illuminating all the preselected points with a single hologram. This process is exemplified for high-speed (up to 1 kHz) two-photon calcium imaging on acute cerebellar slices.

A novel Ca²âº-mediated cross-talk between endoplasmic reticulum and acidic organelles: implications for NAADP-dependent Ca²âº signalling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as the ideal trigger of spatio-temporally complex intracellular Ca(2+) signals. However, the identity of the intracellular Ca(2+) store(s) recruited by NAADP, which may include either the endolysosomal (EL) or the endoplasmic reticulum (ER) Ca(2+) pools, is still elusive. Here, we show that the Ca(2+) response to NAADP was suppressed by interfering with either EL or ER Ca(2+) sequestration. The measurement of EL and ER Ca(2+) levels by using selectively targeted aequorin unveiled that the preventing ER Ca(2+) storage also affected ER Ca(2+) loading and vice versa. This indicates that a functional Ca(2+)-mediated cross-talk exists at the EL-ER interface and exerts profound implications for the study of NAADP-induced Ca(2+) signals. Extreme caution is warranted when dissecting NAADP targets by pharmacologically inhibiting EL and/or the ER Ca(2+) pools. Moreover, Ca(2+) transfer between these compartments might be essential to regulate vital Ca(2+)-dependent processes in both organelles.

The spatiotemporal organization of cerebellar network activity resolved by two-photon imaging of multiple single neurons.

In order to investigate the spatiotemporal organization of neuronal activity in local microcircuits, techniques allowing the simultaneous recording from multiple single neurons are required. To this end, we implemented an advanced spatial-light modulator two-photon microscope (SLM-2PM). A critical issue for cerebellar theory is the organization of granular layer activity in the cerebellum, which has been predicted by single-cell recordings and computational models. With SLM-2PM, calcium signals could be recorded from different network elements in acute cerebellar slices including granule cells (GrCs), Purkinje cells (PCs) and molecular layer interneurons. By combining WCRs with SLM-2PM, the spike/calcium relationship in GrCs and PCs could be extrapolated toward the detection of single spikes. The SLM-2PM technique made it possible to monitor activity of over tens to hundreds neurons simultaneously. GrC activity depended on the number of spikes in the input mossy fiber bursts. PC and molecular layer interneuron activity paralleled that in the underlying GrC population revealing the spread of activity through the cerebellar cortical network. Moreover, circuit activity was increased by the GABA-A receptor blocker, gabazine, and reduced by the AMPA and NMDA receptor blockers, NBQX and APV. The SLM-2PM analysis of spatiotemporal patterns lent experimental support to the time-window and center-surround organizing principles of the granular layer.