Identification and Organization of a Postural Anti-Gravity Module in the Cerebellar Vermis.

The cerebellum is known to control the proper balance of isometric muscular contractions that maintain body posture. Current optogenetic manipulations of the cerebellar cortex output, however, have focused on ballistic body movements, examining movement initiation or perturbations. Here, by optogenetic stimulations of cerebellar Purkinje cells, which are the output of the cerebellar cortex, we evaluate body posture maintenance. By sequential analysis of body movement, we dissect the effect of optogenetic stimulation into a directly induced movement that is then followed by a compensatory reflex to regain body posture. We identify a module in the medial part of the anterior vermis which, through multiple muscle tone regulation, is involved in postural anti-gravity maintenance of the body. Moreover, we report an antero-posterior and medio-lateral functional segregation over the vermal lobules IV/V/VI. Taken together our results open new avenues for better understanding of the modular functional organization of the cerebellar cortex and its role in postural anti-gravity maintenance.

Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.

Fast optical recording of neuronal activity by three-dimensional custom-access serial holography.

Optical recording of neuronal activity in three-dimensional (3D) brain circuits at cellular and millisecond resolution in vivo is essential for probing information flow in the brain. While random-access multiphoton microscopy permits fast optical access to neuronal targets in three dimensions, the method is challenged by motion artifacts when recording from behaving animals. Therefore, we developed three-dimensional custom-access serial holography (3D-CASH). Built on a fast acousto-optic light modulator, 3D-CASH performs serial sampling at 40 kHz from neurons at freely selectable 3D locations. Motion artifacts are eliminated by targeting each neuron with a size-optimized pattern of excitation light covering the cell body and its anticipated displacement field. Spike rates inferred from GCaMP6f recordings in visual cortex of awake mice tracked the phase of a moving bar stimulus with higher spike correlation between intra compared to interlaminar neuron pairs. 3D-CASH offers access to the millisecond correlation structure of in vivo neuronal activity in 3D microcircuits.

Neurotransmitter content heterogeneity within an interneuron class shapes inhibitory transmission at a central synapse.

Neurotransmitter content is deemed the most basic defining criterion for neuronal classes, contrasting with the intercellular heterogeneity of many other molecular and functional features. Here we show, in the adult mouse brain, that neurotransmitter content variegation within a neuronal class is a component of its functional heterogeneity. Golgi cells (GoCs), the well-defined class of cerebellar interneurons inhibiting granule cells (GrCs), contain cytosolic glycine, accumulated by the neuronal transporter GlyT2, and GABA in various proportions. By performing acute manipulations of cytosolic GABA and glycine supply, we find that competition of glycine with GABA reduces the charge of IPSC evoked in GrCs and, more specifically, the amplitude of a slow component of the IPSC decay. We then pair GrCs recordings with optogenetic stimulations of single GoCs, which preserve the intracellular transmitter mixed content. We show that the strength and decay kinetics of GrCs IPSCs, which are entirely mediated by GABAA receptors, are negatively correlated to the presynaptic expression of GlyT2 by GoCs. We isolate a slow spillover component of GrCs inhibition that is also affected by the expression of GlyT2, leading to a 56% decrease in relative charge. Our results support the hypothesis that presynaptic loading of glycine negatively impacts the GABAergic transmission in mixed interneurons, most likely through a competition for vesicular filling. We discuss how the heterogeneity of neurotransmitter supply within mixed interneurons like the GoC class may provide a presynaptic mechanism to tune the gain of microcircuits such as the granular layer, thereby expanding the realm of their possible dynamic behaviors.

Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

Optogenetic stimulation of complex spatio-temporal activity patterns by acousto-optic light steering probes cerebellar granular layer integrative properties.

Optogenetics provides tools to control afferent activity in brain microcircuits. However, this requires optical methods that can evoke asynchronous and coordinated activity within neuronal ensembles in a spatio-temporally precise way. Here we describe a light patterning method, which combines MHz acousto-optic beam steering and adjustable low numerical aperture Gaussian beams, to achieve fast 2D targeting in scattering tissue. Using mossy fiber afferents to the cerebellar cortex as a testbed, we demonstrate single fiber optogenetic stimulation with micron-scale lateral resolution, >100 µm depth-penetration and 0.1 ms spiking precision. Protracted spatio-temporal patterns of light delivered by our illumination system evoked sustained asynchronous mossy fiber activity with excellent repeatability. Combining optical and electrical stimulations, we show that the cerebellar granular layer performs nonlinear integration, whereby sustained mossy fiber activity provides a permissive context for the transmission of salient inputs, enriching combinatorial views on mossy fiber pattern separation.

Voltage- and Branch-Specific Climbing Fiber Responses in Purkinje Cells.

Climbing fibers (CFs) provide instructive signals driving cerebellar learning, but mechanisms causing the variable CF responses in Purkinje cells (PCs) are not fully understood. Using a new experimentally validated PC model, we unveil the ionic mechanisms underlying CF-evoked distinct spike waveforms on different parts of the PC. We demonstrate that voltage can gate both the amplitude and the spatial range of CF-evoked Ca2+ influx by the availability of K+ currents. This makes the energy consumed during a complex spike (CS) also voltage dependent. PC dendrites exhibit inhomogeneous excitability with individual branches as computational units for CF input. The variability of somatic CSs can be explained by voltage state, CF activation phase, and instantaneous CF firing rate. Concurrent clustered synaptic inputs affect CSs by modulating dendritic responses in a spatially precise way. The voltage- and branch-specific CF responses can increase dendritic computational capacity and enable PCs to actively integrate CF signals.

Whole-GUV patch-clamping.

Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

Mechanisms and functional roles of glutamatergic synapse diversity in a cerebellar circuit.

Synaptic currents display a large degree of heterogeneity of their temporal characteristics, but the functional role of such heterogeneities remains unknown. We investigated in rat cerebellar slices synaptic currents in Unipolar Brush Cells (UBCs), which generate intrinsic mossy fibers relaying vestibular inputs to the cerebellar cortex. We show that UBCs respond to sinusoidal modulations of their sensory input with heterogeneous amplitudes and phase shifts. Experiments and modeling indicate that this variability results both from the kinetics of synaptic glutamate transients and from the diversity of postsynaptic receptors. While phase inversion is produced by an mGluR2-activated outward conductance in OFF-UBCs, the phase delay of ON UBCs is caused by a late rebound current resulting from AMPAR recovery from desensitization. Granular layer network modeling indicates that phase dispersion of UBC responses generates diverse phase coding in the granule cell population, allowing climbing-fiber-driven Purkinje cell learning at arbitrary phases of the vestibular input.

Burst-Dependent Bidirectional Plasticity in the Cerebellum Is Driven by Presynaptic NMDA Receptors.

Numerous studies have shown that cerebellar function is related to the plasticity at the synapses between parallel fibers and Purkinje cells. How specific input patterns determine plasticity outcomes, as well as the biophysics underlying plasticity of these synapses, remain unclear. Here, we characterize the patterns of activity that lead to postsynaptically expressed LTP using both in vivo and in vitro experiments. Similar to the requirements of LTD, we find that high-frequency bursts are necessary to trigger LTP and that this burst-dependent plasticity depends on presynaptic NMDA receptors and nitric oxide (NO) signaling. We provide direct evidence for calcium entry through presynaptic NMDA receptors in a subpopulation of parallel fiber varicosities. Finally, we develop and experimentally verify a mechanistic plasticity model based on NO and calcium signaling. The model reproduces plasticity outcomes from data and predicts the effect of arbitrary patterns of synaptic inputs on Purkinje cells, thereby providing a unified description of plasticity.

Fast spatial beam shaping by acousto-optic diffraction for 3D non-linear microscopy.

Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions.

A novel inhibitory nucleo-cortical circuit controls cerebellar Golgi cell activity.

The cerebellum, a crucial center for motor coordination, is composed of a cortex and several nuclei. The main mode of interaction between these two parts is considered to be formed by the inhibitory control of the nuclei by cortical Purkinje neurons. We now amend this view by showing that inhibitory GABA-glycinergic neurons of the cerebellar nuclei (CN) project profusely into the cerebellar cortex, where they make synaptic contacts on a GABAergic subpopulation of cerebellar Golgi cells. These spontaneously firing Golgi cells are inhibited by optogenetic activation of the inhibitory nucleo-cortical fibers both in vitro and in vivo. Our data suggest that the CN may contribute to the functional recruitment of the cerebellar cortex by decreasing Golgi cell inhibition onto granule cells.

CaRuby-Nano: a novel high affinity calcium probe for dual color imaging.

The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

A subcortical inhibitory signal for behavioral arrest in the thalamus.

Organization of behavior requires rapid coordination of brainstem and forebrain activity. The exact mechanisms of effective communication between these regions are presently unclear. The intralaminar thalamic nuclei (IL) probably serves as a central hub in this circuit by connecting the critical brainstem and forebrain areas. We found that GABAergic and glycinergic fibers ascending from the pontine reticular formation (PRF) of the brainstem evoked fast and reliable inhibition in the IL via large, multisynaptic terminals. This inhibition was fine-tuned through heterogeneous GABAergic and glycinergic receptor ratios expressed at individual synapses. Optogenetic activation of PRF axons in the IL of freely moving mice led to behavioral arrest and transient interruption of awake cortical activity. An afferent system with comparable morphological features was also found in the human IL. These data reveal an evolutionarily conserved ascending system that gates forebrain activity through fast and powerful synaptic inhibition of the IL.

Activity-dependent gating of calcium spikes by A-type K+ channels controls climbing fiber signaling in Purkinje cell dendrites.

In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules.

Differential GABAergic and glycinergic inputs of inhibitory interneurons and Purkinje cells to principal cells of the cerebellar nuclei.

The principal neurons of the cerebellar nuclei (CN), the sole output of the olivo-cerebellar system, receive a massive inhibitory input from Purkinje cells (PCs) of the cerebellar cortex. Morphological evidence suggests that CN principal cells are also contacted by inhibitory interneurons, but the properties of this connection are unknown. Using transgenic, tracing, and immunohistochemical approaches in mice, we show that CN interneurons form a large heterogeneous population with GABA/glycinergic phenotypes, distinct from GABAergic olive-projecting neurons. CN interneurons are found to contact principal output neurons, via glycine receptor (GlyR)-enriched synapses, virtually devoid of the main GABA receptor (GABAR) subunits α1 and γ2. Those clusters account for 5% of the total number of inhibitory receptor clusters on principal neurons. Brief optogenetic stimulations of CN interneurons, through selective expression of channelrhodopsin 2 after viral-mediated transfection of the flexed gene in GlyT2-Cre transgenic mice, evoked fast IPSCs in principal cells. GlyR activation accounted for 15% of interneuron IPSC amplitude, while the remaining current was mediated by activation of GABAR. Surprisingly, small GlyR clusters were also found at PC synapses onto principal CN neurons in addition to α1 and γ2 GABAR subunits. However, GlyR activation was found to account for <3% of the PC inhibitory synaptic currents evoked by electrical stimulation. This work establishes CN glycinergic neurons as a significant source of inhibition to CN principal cells, forming contacts molecularly distinct from, but functionally similar to, Purkinje cell synapses. Their impact on CN output, motor learning, and motor execution deserves further investigation.

Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge.

Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning.

Granule cell ascending axon excitatory synapses onto Golgi cells implement a potent feedback circuit in the cerebellar granular layer.

The function of inhibitory interneurons within brain microcircuits depends critically on the nature and properties of their excitatory synaptic drive. Golgi cells (GoCs) of the cerebellum inhibit cerebellar granule cells (GrCs) and are driven both by feedforward mossy fiber (mf) and feedback GrC excitation. Here, we have characterized GrC inputs to GoCs in rats and mice. We show that, during sustained mf discharge, synapses from local GrCs contribute equivalent charge to GoCs as mf synapses, arguing for the importance of the feedback inhibition. Previous studies predicted that GrC-GoC synapses occur predominantly between parallel fibers (pfs) and apical GoC dendrites in the molecular layer (ML). By combining EM and Ca(2+) imaging, we now demonstrate the presence of functional synaptic contacts between ascending axons (aa) of GrCs and basolateral dendrites of GoCs in the granular layer (GL). Immunohistochemical quantification estimates these contacts to be ∼400 per GoC. Using Ca(2+) imaging to identify synaptic inputs, we show that EPSCs from aa and mf contacts in basolateral dendrites display similarly fast kinetics, whereas pf inputs in the ML exhibit markedly slower kinetics as they undergo strong filtering by apical dendrites. We estimate that approximately half of the local GrC contacts generate fast EPSCs, indicating their basolateral location in the GL. We conclude that GrCs, through their aa contacts onto proximal GoC dendrites, define a powerful feedback inhibitory circuit in the GL.

NMDA receptors with incomplete Mg²âº block enable low-frequency transmission through the cerebellar cortex.

The cerebellar cortex coordinates movements and maintains balance by modifying motor commands as a function of sensory-motor context, which is encoded by mossy fiber (MF) activity. MFs exhibit a wide range of activity, from brief precisely timed high-frequency bursts, which encode discrete variables such as whisker stimulation, to low-frequency sustained rate-coded modulation, which encodes continuous variables such as head velocity. While high-frequency MF inputs have been shown to activate granule cells (GCs) effectively, much less is known about sustained low-frequency signaling through the GC layer, which is impeded by a hyperpolarized resting potential and strong GABA(A)-mediated tonic inhibition of GCs. Here we have exploited the intrinsic MF network of unipolar brush cells to activate GCs with sustained low-frequency asynchronous MF inputs in rat cerebellar slices. We find that low-frequency MF input modulates the intrinsic firing of Purkinje cells, and that this signal transmission through the GC layer requires synaptic activation of Mg²âº-block-resistant NMDA receptors (NMDARs) that are likely to contain the GluN2C subunit. Slow NMDAR conductances sum temporally to contribute approximately half the MF-GC synaptic charge at hyperpolarized potentials. Simulations of synaptic integration in GCs show that the NMDAR and slow spillover-activated AMPA receptor (AMPAR) components depolarize GCs to a similar extent. Moreover, their combined depolarizing effect enables the fast quantal AMPAR component to trigger action potentials at low MF input frequencies. Our results suggest that the weak Mg²âº block of GluN2C-containing NMDARs enables transmission of low-frequency MF signals through the input layer of the cerebellar cortex.