Cerebellum Lecture: the Cerebellar Nuclei-Core of the Cerebellum.

The cerebellum is a key player in many brain functions and a major topic of neuroscience research. However, the cerebellar nuclei (CN), the main output structures of the cerebellum, are often overlooked. This neglect is because research on the cerebellum typically focuses on the cortex and tends to treat the CN as relatively simple output nuclei conveying an inverted signal from the cerebellar cortex to the rest of the brain. In this review, by adopting a nucleocentric perspective we aim to rectify this impression. First, we describe CN anatomy and modularity and comprehensively integrate CN architecture with its highly organized but complex afferent and efferent connectivity. This is followed by a novel classification of the specific neuronal classes the CN comprise and speculate on the implications of CN structure and physiology for our understanding of adult cerebellar function. Based on this thorough review of the adult literature we provide a comprehensive overview of CN embryonic development and, by comparing cerebellar structures in various chordate clades, propose an interpretation of CN evolution. Despite their critical importance in cerebellar function, from a clinical perspective intriguingly few, if any, neurological disorders appear to primarily affect the CN. To highlight this curious anomaly, and encourage future nucleocentric interpretations, we build on our review to provide a brief overview of the various syndromes in which the CN are currently implicated. Finally, we summarize the specific perspectives that a nucleocentric view of the cerebellum brings, move major outstanding issues in CN biology to the limelight, and provide a roadmap to the key questions that need to be answered in order to create a comprehensive integrated model of CN structure, function, development, and evolution.

Diversity of neuronal elements and circuitry in the cerebellar nuclei.

The afferent and efferent synaptic connections of the cerebellar nuclei (CN) place them in a key position where they can integrate sensory signals with the output from cerebellar cortex and to provide the main efferent pathway of the cerebellum. While this conclusion can be derived based on purely anatomical knowledge, it remains unknown in which manner the CN contributes to the generation of cerebellar output signals that are involved in creating timing signals and temporal patterns. As a first step towards understanding the role neuronal circuits of the CN, the major CN neuronal types are now identified based on expression patterns of neurotransmitters (GABA and glycine) and characterized both in electrophysiological and morphological manner. The classification-likely to be refined in the future-consists of six types: four classes of projection and two classes of local neurons. The classification is a combination of electrophysiological and morphological methods with the expression pattern of GAD67 and GlyT2, markers for GABAergic and glycinergic neurons, respectively (Uusisaari et al. J Neurophysiol 97:901-911, 2007; Uusisaari and Knöpfel, Cerebellum 10(4):637-46, 2010).

Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo.

Adeno-associated viral (AAV) vectors, used as vehicles for gene transfer into the brain, are a versatile and powerful tool of modern neuroscience that allow identifying specific neuronal populations, monitoring and modulating their activity. For consistent and reproducible results, the AAV vectors must be engineered so that they reliably and accurately target cell populations. Furthermore, transgene expression must be adjusted to sufficient and safe levels compatible with the physiology of studied cells. We undertook the effort to identify and validate an AAV vector that could be utilized for researching the inferior olivary (IO) nucleus, a structure gating critical timing-related signals to the cerebellum. By means of systematic construct generation and quantitative expression profiling, we succeeded in creating a viral tool for specific and strong transfection of the IO neurons without adverse effects on their physiology. The potential of these tools is demonstrated by expressing the calcium sensor GCaMP6s in adult mouse IO neurons. We could monitor subtle calcium fluctuations underlying two signatures of intrinsic IO activity: the subthreshold oscillations (STOs) and the variable-duration action potential waveforms both in-vitro and in-vivo. Further, we show that the expression levels of GCaMP6s allowing such recordings are compatible with the delicate calcium-based dynamics of IO neurons, inviting future work into the network dynamics of the olivo-cerebellar system in behaving animals.

In vivo Calcium Imaging in Mouse Inferior Olive.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest. Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro.

Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period in vivo in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.

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.

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.

Cerebellar inhibitory input to the inferior olive decreases electrical coupling and blocks subthreshold oscillations.

GABAergic projection neurons in the cerebellar nuclei (CN) innervate the inferior olive (IO) that in turn is the source of climbing fibers targeting Purkinje neurons in the cerebellar cortex. Anatomical evidence suggests that CN synapses modulate electrical coupling between IO neurons. In vivo studies indicate that they are also involved in controlling synchrony and rhythmicity of IO neurons. Here, we demonstrate using virally targeted channelrhodopsin in the cerebellar nucleo-olivary neurons that synaptic input can indeed modulate both the strength and symmetry of electrical coupling between IO neurons and alter network activity. Similar synaptic modifications of electrical coupling are likely to occur in other brain regions, where rapid modification of the spatiotemporal features of the coupled networks is needed to adequately respond to behavioral demands.