How inhibitory and excitatory inputs gate output of the inferior olive.

The inferior olive provides the climbing fibers to Purkinje cells in the cerebellar cortex, where they elicit all-or-none complex spikes and control major forms of plasticity. Given their important role in both short-term and long-term coordination of cerebellum-dependent behaviors, it is paramount to understand the factors that determine the output of olivary neurons. Here, we use mouse models to investigate how the inhibitory and excitatory inputs to the olivary neurons interact with each other, generating spiking patterns of olivary neurons that align with their intrinsic oscillations.

Purkinje cells translate subjective salience into readiness to act and choice performance.

The brain selectively allocates attention from a continuous stream of sensory input. This process is typically attributed to computations in distinct regions of the forebrain and midbrain. Here, we explore whether cerebellar Purkinje cells encode information about the selection of sensory inputs and could thereby contribute to non-motor forms of learning.

OptiFlex: Multi-Frame Animal Pose Estimation Combining Deep Learning With Optical Flow.

Animal pose estimation tools based on deep learning have greatly improved animal behaviour quantification. These tools perform pose estimation on individual video frames, but do not account for variability of animal body shape in their prediction and evaluation. Here, we introduce a novel multi-frame animal pose estimation framework, referred to as OptiFlex.

NINscope, a versatile miniscope for multi-region circuit investigations.

Miniaturized fluorescence microscopes (miniscopes) have been instrumental to monitor neural signals during unrestrained behavior and their open-source versions have made them affordable. Often, the footprint and weight of open-source miniscopes is sacrificed for added functionality. Here, we present NINscope: a light-weight miniscope with a small footprint that integrates a high-sensitivity image sensor, an inertial measurement unit and an LED driver for an external optogenetic probe.

Circuit Investigations With Open-Source Miniaturized Microscopes: Past, Present and Future.

The ability to simultaneously image the spatiotemporal activity signatures from many neurons during unrestrained vertebrate behaviors has become possible through the development of miniaturized fluorescence microscopes, or miniscopes, sufficiently light to be carried by small animals such as bats, birds and rodents. Miniscopes have permitted the study of circuits underlying song vocalization, action sequencing, head-direction tuning, spatial memory encoding and sleep to name a few. The foundation for these microscopes has been laid over the last two decades through academic research with some of this work resulting in commercialization.

Variability and directionality of inferior olive neuron dendrites revealed by detailed 3D characterization of an extensive morphological library.

The inferior olive (IO) is an evolutionarily conserved brain stem structure and its output activity plays a major role in the cerebellar computation necessary for controlling the temporal accuracy of motor behavior. The precise timing and synchronization of IO network activity has been attributed to the dendro-dendritic gap junctions mediating electrical coupling within the IO nucleus. Thus, the dendritic morphology and spatial arrangement of IO neurons governs how synchronized activity emerges in this nucleus.

Reappraisal of Bergmann glial cells as modulators of cerebellar circuit function.

Just as there is a huge morphological and functional diversity of neuron types specialized for specific aspects of information processing in the brain, astrocytes have equally distinct morphologies and functions that aid optimal functioning of the circuits in which they are embedded. One type of astrocyte, the Bergmann glial cell (BG) of the cerebellum, is a prime example of a highly diversified astrocyte type, the architecture of which is adapted to the cerebellar circuit and facilitates an impressive range of functions that optimize information processing in the adult brain. In this review we expand on the function of the BG in the cerebellum to highlight the importance of astrocytes not only in housekeeping functions, but also in contributing to plasticity and information processing in the cerebellum.

Role of Synchronous Activation of Cerebellar Purkinje Cell Ensembles in Multi-joint Movement Control.

It is a longstanding question in neuroscience how elaborate multi-joint movements are coordinated coherently. Microzones of cerebellar Purkinje cells (PCs) are thought to mediate this coordination by controlling the timing of particular motor domains. However, it remains to be elucidated to what extent motor coordination deficits can be correlated with abnormalities in coherent activity within these microzones and to what extent artificially evoked synchronous activity within PC ensembles can elicit multi-joint motor behavior.

Behavioral correlates of complex spike synchrony in cerebellar microzones.

The olivo-cerebellar system is crucial for smooth and well timed execution of movements based on sensory and proprioceptive cues. The inferior olive (IO) plays a pivotal role in this process by synchronizing its activity across neurons internally through connexin36 gap junctions and providing a timing and/or learning signal to the cerebellum. Even though synchrony achieved through electrical coupling in IO cells is generally thought to be important in timing motor output, a direct relation between timing of movement and synchrony of olivary discharges has never been demonstrated within functional microcomplexes using transgenics.

Strength and timing of motor responses mediated by rebound firing in the cerebellar nuclei after Purkinje cell activation.

The cerebellum refines the accuracy and timing of motor performance. How it encodes information to perform these functions is a major topic of interest. We performed whole cell and extracellular recordings of Purkinje cells (PCs) and cerebellar nuclei neurons (CNs) in vivo, while activating PCs with light in transgenic mice.

Widespread state-dependent shifts in cerebellar activity in locomoting mice.

Excitatory drive enters the cerebellum via mossy fibers, which activate granule cells, and climbing fibers, which activate Purkinje cell dendrites. Until now, the coordinated regulation of these pathways has gone unmonitored in spatially resolved neuronal ensembles, especially in awake animals. We imaged cerebellar activity using functional two-photon microscopy and extracellular recording in awake mice locomoting on an air-cushioned spherical treadmill.

A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.

Fluorescent calcium indicator proteins, such as GCaMP3, allow imaging of activity in genetically defined neuronal populations. GCaMP3 can be expressed using various gene delivery methods, such as viral infection or electroporation. However, these methods are invasive and provide inhomogeneous and nonstationary expression.

Activity-dependent increases in local oxygen consumption correlate with postsynaptic currents in the mouse cerebellum in vivo.

Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO(2)) and cerebral blood flow (CBF). Activity-dependent rises in CMRO(2) fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca(2+) stimulate oxidative metabolism via mitochondrial signaling, but whether this also occurs in the intact brain is unknown.

Preferential loading of bergmann glia with synthetic acetoxymethyl calcium dyes.

The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. This protocol outlines a method for in vivo calcium imaging in cerebellar astrocytes of mice which have undergone a cerebellar craniotomy.

Cerebellar craniotomy for in vivo calcium imaging of astrocytes.

The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. It is possible to perform in vivo calcium imaging in cerebellar astrocytes.

Injection of recombinant adenovirus for delivery of genetically encoded calcium indicators into astrocytes of the cerebellar cortex.

The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. This protocol outlines a method for in vivo calcium imaging in cerebellar astrocytes, using the injection of a replication-incompetent recombinant adenovirus for gene transfer of the fluorescent calcium indicator protein (FCIP) G-CaMP2.

Recent developments in the understanding of astrocyte function in the cerebellum in vivo.

Several studies have contributed to our understanding of astrocytes, especially Bergmann glia, in the cerebellum; but, until recently, none has looked at their function in vivo. Multicell bolus loading of fluorescent calcium indicators in combination with the astrocytic marker SR101 has allowed imaging of up to hundreds of astrocytes at once in the intact cerebellum. In addition, the selective targeting of astrocytes with fluorescent calcium indicator proteins has enabled the study of their function in vivo without the confounding effects of other neuropil signals and with a resolution that surpasses multicell bolus loading and SR101 staining.

Radially expanding transglial calcium waves in the intact cerebellum.

Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent indicator dyes or the calcium-sensor protein G-CaMP2, we discovered spontaneous calcium waves that filled approximately ellipsoidal domains of Bergmann glia processes.

Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy.

The successful study of dendritic signaling and computation requires the ability to simultaneously monitor neuronal activity at multiple cellular sites. While the difficulties of accessing dendritic submicron structures with conventional micropipette approaches are generally overcome by optical recording techniques, their spatio-temporal resolution has limited such studies to few sites or slow signals. Here we present a novel approach to functional imaging, termed random-access multiphoton (RAMP) microscopy, which combines multiphoton excitation with an inertia-free scanning mechanism.