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. More recently, open-source initiatives have led to an even broader adoption of miniscopes in the neuroscience community. Open-source designs allow for rapid modification and extension of their function, which has resulted in a new generation of miniscopes that now permit wire-free or wireless recording, concurrent electrophysiology and imaging, two-color fluorescence detection, simultaneous optical actuation and read-out as well as wide-field and volumetric light-field imaging. These novel miniscopes will further expand the toolset of those seeking affordable methods to probe neural circuit function during naturalistic behaviors. Here, we will discuss the early development, present use and future potential of miniscopes

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. Using dual color optogenetic stimulation and whole-cell recordings, we demonstrate how intervals between the inhibitory input from the cerebellar nuclei and excitatory input from the mesodiencephalic junction affect phase and gain of the olivary output at both the sub- and suprathreshold level. When the excitatory input is activated shortly (~50 ms) after the inhibitory input, the phase of the intrinsic oscillations becomes remarkably unstable and the excitatory input can hardly generate any olivary spike. Instead, when the excitatory input is activated one cycle (~150 ms) after the inhibitory input, the excitatory input can optimally drive olivary spiking, riding on top of the first cycle of the subthreshold oscillations that have been powerfully reset by the preceding inhibitory input. Simulations of a large-scale network model of the inferior olive highlight to what extent the synaptic interactions penetrate in the neuropil, generating quasi-oscillatory spiking patterns in large parts of the olivary subnuclei, the size of which also depends on the relative timing of the inhibitory and excitatory inputs.</p

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. Using dual color optogenetic stimulation and whole-cell recordings, we demonstrate how intervals between the inhibitory input from the cerebellar nuclei and excitatory input from the mesodiencephalic junction affect phase and gain of the olivary output at both the sub- and suprathreshold level. When the excitatory input is activated shortly (~50 ms) after the inhibitory input, the phase of the intrinsic oscillations becomes remarkably unstable and the excitatory input can hardly generate any olivary spike. Instead, when the excitatory input is activated one cycle (~150 ms) after the inhibitory input, the excitatory input can optimally drive olivary spiking, riding on top of the first cycle of the subthreshold oscillations that have been powerfully reset by the preceding inhibitory input. Simulations of a large-scale network model of the inferior olive highlight to what extent the synaptic interactions penetrate in the neuropil, generating quasi-oscillatory spiking patterns in large parts of the olivary subnuclei, the size of which also depends on the relative timing of the inhibitory and excitatory inputs.</p

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. We use it to perform the first concurrent cellular resolution recordings from cerebellum and cerebral cortex in unrestrained mice, demonstrate its optogenetic stimulation capabilities to examine cerebello-cerebral or cortico-striatal connectivity, and replicate findings of action encoding in dorsal striatum. In combination with cross-platform acquisition and control software, our miniscope is a versatile addition to the expanding tool chest of open-source miniscopes that will increase access to multi-region circuit investigations during unrestrained behavior

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. To date, IO neuron structural properties have been characterized in few studies and with small numbers of neurons; these investigations have described IO neurons as belonging to two morphologically distinct types, “curly” and “straight”. In this work we collect a large number of individual IO neuron morphologies visualized using different labeling techniques and present a thorough examination of their morphological properties and spatial arrangement within the olivary neuropil. Our results show that the extensive heterogeneity in IO neuron dendritic morphologies occupies a continuous range between the classically described “curly” and “straight” types, and that this continuum is well represented by a relatively simple measure of “straightness”. Furthermore, we find that IO neuron dendritic trees are often directionally oriented. Combined with an examination of cell body density distributions and dendritic orientation of adjacent IO neurons, our results suggest that the IO network may be organized into groups of densely coupled neurons interspersed with areas of weaker coupling

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 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 Bergmann glial cell 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

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. Here we combined in vivo, two-photon calcium imaging of complex spikes in microcomplexes of Purkinje cell (PC) dendrites with high-speed filming of tail, trunk, and limb movements in awake wild-type and connexin36-deficient mice. In wild types at rest, functional clusters of PCs were poorly defined with synchrony correlations that were relatively small and spatially limited to mediolateral distances of ∼50 μm, whereas during locomotion synchrony of the same PCs increased in strength and extended over distances spanning multiple microzones that could be correlated to specific components of sharp and well bounded movements. Instead, connexin36-deficient mice exhibited prolonged and desynchronized complex spike activity within PC microcomplexes both at rest and during behavior. Importantly, the mutants also showed concomitant abnormalities in the execution of spinocerebellar reflexes, which were significantly slower and more gradual than in wild-type littermates, particularly following sensory perturbations. Our results highlight the importance of modulation of synchronous activity within and between cerebellar microcomplexes in on-line temporal processing of motor output