Olov Oscarsson's Description of Afferent Pathways to the Cerebellum: Excellent Physiology, Base for Anatomy, and Road Toward Understanding Function.

Olov Oscarsson's review on the functional organization of spinocerebellar paths is a prime demonstration of the great skills and huge knowledge base of the electrophysiologists of his era working on communication systems in the brain. Oscarsson describes and characterizes in detail no less than ten different communication lines between the spinal cord and the cerebellum. As such, his work proved to be a highly fertile basis for ongoing physiological and anatomical research. However, even after 50 years of continuing cerebellar research, many questions are still open and even care must be taken that the differentiation in spinocerebellar paths, so carefully demonstrated by Oscarsson, is not lost in present-day research.

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.

Input and output organization of the mesodiencephalic junction for cerebro-cerebellar communication.

Most studies investigating the impact of the cerebral cortex (CC) onto the cerebellum highlight the role of the pons, which provides the mossy fibers to the cerebellum. However, cerebro-cerebellar communication may also be mediated by the nuclei of the mesodiencephalic junction (MDJ) that project to the inferior olive (IO), which in turn provides the climbing fibers to the molecular layer. Here, we uncover the precise topographic relations of the inputs and outputs of the MDJ using multiple, classical, and transneuronal tracing methods as well as analyses of mesoscale cortical injections from Allen Mouse Brain. We show that the caudal parts of the CC predominantly project to the principal olive via the rostral MDJ and that the rostral parts of the CC predominantly project to the rostral medial accessory olive via the caudal MDJ. Moreover, using triple viral tracing technology, we show that the cerebellar nuclei directly innervate the neurons in the MDJ that receive input from CC and project to the IO. By unraveling these topographic and prominent, mono- and disynaptic projections through the MDJ, this work establishes that cerebro-cerebellar communication is not only mediated by the pontine mossy fiber system, but also by the climbing fiber system.

Multizonal Cerebellar Influence Over Sensorimotor Areas of the Rat Cerebral Cortex.

The cerebral cortex requires cerebellar input for optimizing sensorimotor processing. However, how the sensorimotor cortex uses cerebellar information is far from understood. One critical and unanswered question is how cerebellar functional entities (zones or modules) are connected to distinct parts of the sensorimotor cortices. Here, we utilized retrograde transneuronal infection of rabies virus (RABV) to study the organization of connections from the cerebellar cortex to M1, M2, and S1 of the rat cerebral cortex. RABV was co-injected with cholera toxin ß-subunit (CTb) into each of these cortical regions and a survival time of 66-70 h allowed for third-order retrograde RABV infection of Purkinje cells. CTb served to identify the injection site. RABV+ Purkinje cells throughout cerebellar zones were identified by reference to the cerebellar zebrin pattern. All injections, including those into S1, resulted in multiple, zonally arranged, strips of RABV+ Purkinje cells. M1 injections were characterized by input from Purkinje cells in the vermal X-zone, medial paravermis (C1- and Cx-zones), and lateral hemisphere (D2-zone); M2 receives input from D2- and C3-zones; connections to S1 originate from X-, Cx-, C3-, and D2-zones. We hypothesize that individual domains of the sensorimotor cortex require information from a specific combination of cerebellar modules.

Correction to: Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper.

In the original version of this paper, the Title should have been written with "A Consensus paper" to read "Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper".

Cerebellar Modules and Their Role as Operational Cerebellar Processing Units: A Consensus paper [corrected].

The compartmentalization of the cerebellum into modules is often used to discuss its function. What, exactly, can be considered a module, how do they operate, can they be subdivided and do they act individually or in concert are only some of the key questions discussed in this consensus paper. Experts studying cerebellar compartmentalization give their insights on the structure and function of cerebellar modules, with the aim of providing an up-to-date review of the extensive literature on this subject. Starting with an historical perspective indicating that the basis of the modular organization is formed by matching olivocorticonuclear connectivity, this is followed by consideration of anatomical and chemical modular boundaries, revealing a relation between anatomical, chemical, and physiological borders. In addition, the question is asked what the smallest operational unit of the cerebellum might be. Furthermore, it has become clear that chemical diversity of Purkinje cells also results in diversity of information processing between cerebellar modules. An additional important consideration is the relation between modular compartmentalization and the organization of the mossy fiber system, resulting in the concept of modular plasticity. Finally, examination of cerebellar output patterns suggesting cooperation between modules and recent work on modular aspects of emotional behavior are discussed. Despite the general consensus that the cerebellum has a modular organization, many questions remain. The authors hope that this joint review will inspire future cerebellar research so that we are better able to understand how this brain structure makes its vital contribution to behavior in its most general form.

Visuo-vestibular information processing by unipolar brush cells in the rabbit flocculus.

The unipolar brush cell (UBC) is a glutamatergic granular layer interneuron that is predominantly located in the vestibulocerebellum and parts of the vermis. In rat and rabbit, we previously found using juxtacellular labeling combined with spontaneous activity recording that cells with highly regular spontaneous activity belong to the UBC category. Making use of this signature, we recorded from floccular UBCs in both anesthetized and awake rabbits while delivering visuo-vestibular stimulation by using sigmoidal rotation of the whole animal. In the anesthetized rabbit, the activity of the presumed UBC units displayed a wide variety of modulation profiles that could be related to aspects of head velocity or acceleration. These modulation profiles could also be found in the awake rabbit where, in addition, they could also carry an eye position signal. Furthermore, units in the awake rabbit could demonstrate rather long response latencies of up to 0.5 s. We suggest that the UBCs recorded in this study mostly belong to the type I UBC category (calretinin-positive) and that they can play diverse roles in floccular visuo-vestibular information processing, such as transformation of velocity-related signals to acceleration-related signals.

Axonal sprouting and formation of terminals in the adult cerebellum during associative motor learning.

Plastic changes in the efficacy of synapses are widely regarded to represent mechanisms underlying memory formation. So far, evidence for learning-dependent, new neuronal wiring is limited. In this study, we demonstrate that pavlovian eyeblink conditioning in adult mice can induce robust axonal growth and synapse formation in the cerebellar nuclei. This de novo wiring is both condition specific and region specific because it does not occur in pseudoconditioned animals and is particularly observed in those parts of the cerebellar nuclei that have been implicated to be involved in this form of motor learning. Moreover, the number of new mossy fiber varicosities in these parts of the cerebellar nuclei is positively correlated with the amplitude of conditioned eyelid responses. These results indicate that outgrowth of axons and concomitant occurrence of new terminals may, in addition to plasticity of synaptic efficacy, contribute to the formation of memory.

Organization of cerebral projections to identified cerebellar zones in the posterior cerebellum of the rat.

The cerebrocerebellar connection makes use of two of the largest fiber tracts in the mammalian brain, i.e., the cerebral and medial cerebellar peduncles. Neuroanatomical approaches aimed to elucidate the organization of this important connection have been hindered by its multisynaptic nature, the complex organization of its components, and the dependency of conventional tracers on precisely placed injections. To overcome these problems, we used rabies virus (RV) as a retrograde transneuronal tracer. RV was injected simultaneously with cholera toxin ß subunit (CTb) into selected areas of the cerebellar cortex of 18 male Wistar rats. A survival time of 48-50 h resulted in first- and second-order labeling of RV in combination with first-order labeling of CTb. The distribution of CTb-labeled neurons in the inferior olive established the zonal identity of the injection site. In this way, it was possible to examine the cortical distribution of neurons from which disynaptic cerebrocerebellar projections to specific cerebellar loci originate. The results show that this distribution covaries with the identity of the injected cerebellar lobule. More subtle changes were present when different zones of the same lobule were injected. The C1 zone of lobule VIII receives a more prominent projection from the somatosensory cortex compared with the C2/D zones. The laterally positioned D zones receive information from more rostral regions of the cerebral cortex. The vermis of lobule VII receives a prominent input from the retrosplenial and orbitofrontal cortices. Different injection sites also result in differences in laterality of the connections.

Differential olivo-cerebellar cortical control of rebound activity in the cerebellar nuclei.

The output of the cerebellar cortex is controlled by two main inputs, (i.e., the climbing fiber and mossy fiber-parallel fiber pathway) and activations of these inputs elicit characteristic effects in its Purkinje cells: that is, the so-called complex spikes and simple spikes. Target neurons of the Purkinje cells in the cerebellar nuclei show rebound firing, which has been implicated in the processing and storage of motor coordination signals. Yet, it is not known to what extent these rebound phenomena depend on different modes of Purkinje cell activation. Using extracellular as well as patch-clamp recordings, we show here in both anesthetized and awake rodents that simple and complex spike-like train stimuli to the cerebellar cortex, as well as direct activation of the inferior olive, all result in rebound increases of the firing frequencies of cerebellar nuclei neurons for up to 250 ms, whereas single-pulse stimuli to the cerebellar cortex predominantly elicit well-timed spiking activity without changing the firing frequency of cerebellar nuclei neurons. We conclude that the rebound phenomenon offers a rich and powerful mechanism for cerebellar nuclei neurons, which should allow them to differentially process the climbing fiber and mossy fiber inputs in a physiologically operating cerebellum.

A disynaptic basal ganglia connection to the inferior olive: potential for basal ganglia influence on cerebellar learning.

Recent studies have shown that the cerebellum and the basal ganglia are interconnected at subcortical levels. However, a subcortical basal ganglia connection to the inferior olive (IO), being the source of the olivocerebellar climbing fiber system, is not known. We have used classical tracing with CTb, retrograde transneuronal infection with wildtype rabies virus, conditional tracing with genetically modified rabies virus, and examination of material made available by the Allen Brain Institute, to study potential basal ganglia connections to the inferior olive in rats and mice. We show in both species that parvalbumin-positive, and therefore GABAergic, neurons in the entopeduncular nucleus, representing the rodent equivalent of the internal part of the globus pallidus, innervate a group of cells that surrounds the fasciculus retroflexus and that are collectively known as the area parafascicularis prerubralis. As these neurons supply a direct excitatory input to large parts of the inferior olivary complex, we propose that the entopeduncular nucleus, as a main output station of the basal ganglia, provides an inhibitory influence on olivary excitability. As such, this connection may influence olivary involvement in cerebellar learning and/or could be involved in transmission of reward properties that have recently been established for olivocerebellar signaling.

Sympathetic components in left and right human cervical vagus nerve: implications for vagus nerve stimulation.

Cervical vagus nerve stimulation is in a great variety of clinical situations indicated as a form of treatment. It is textbook knowledge that at the cervical level the vagus nerve contains many different fiber classes. Yet, recently, several reports have shown that this nerve also may contain an additional class of potentially noradrenergic fibers, suggested to denote efferent sympathetic fibers. As such, the nature and presence of these fibers should be considered when choosing a stimulation protocol. We have studied human vagus material extracted from dissection room cadavers in order to further confirm the presence of this class of fibers, to study their origin and direction within the nerve and to determine their distribution and variability between subjects and pairs of left and right nerves of the same individual. Sections were studied with immunohistochemical techniques using antibodies against tyrosine hydroxylase (TH: presumed to indicate noradrenergic fibers), myelin basic protein and neurofilament. Our results show that at least part of the TH-positive fibers derive from the superior cervical ganglion or sympathetic trunk, do not follow a cranial but take a peripheral course through the nerve. The portion of TH-positive fibers is highly variable between individuals but also between the left and right pairs of the same individual. TH-positive fibers can distribute and wander throughout the fascicles but maintain a generally clustered appearance. The fraction of TH-positive fibers generally diminishes in the left cervical vagus nerve when moving in a caudal direction but remains more constant in the right nerve. These results may help to determine optimal stimulation parameters for cervical vagus stimulation in clinical settings.

Spontaneous activity signatures of morphologically identified interneurons in the vestibulocerebellum.

Cerebellar cortical interneurons such as Golgi cells, basket cells, stellate cells, unipolar brush cells, and granule cells play an essential role in the operations of the cerebellum. However, detailed functional studies of the activity of these cells in both anesthetized and behaving animals have been hampered by problems in recognizing their physiological signatures. We have extracellularly recorded the spontaneous activity of vestibulocerebellar interneurons in ketamine/xylazine-anesthetized rats and subsequently labeled them with Neurobiotin using the juxtacellular technique. After recovery and morphological identification of these cells, they were related to statistical measures of their spontaneous activity. Golgi cells display a somewhat irregular firing pattern with relatively low average frequencies. Unipolar brush cells are characterized by more regular firing at higher rates. Basket and stellate cells are alike in their firing characteristics, which mainly stand out by their irregularity; some of them are set apart by their very slow average rate. The spontaneous activity of interneurons examined in the ketamine/xylazine rabbit fit within this general pattern. In the rabbit, granule cells were identified by the spontaneous occurrence of extremely high-frequency bursts of action potentials, which were also recognized in the rat. On the basis of these observations, we devised an algorithm that reliably determined the identity of 75% of the cells with only 2% incorrect classifications. The remaining cells were placed into border categories within which no classification was attempted. We propose that this algorithm can be used to help classify vestibulocerebellar interneurons recorded in awake, behaving animals.

Spatiotemporal dynamics of re-innervation and hyperinnervation patterns by uninjured CGRP fibers in the rat foot sole epidermis after nerve injury.

The epidermis is innervated by fine nerve endings that are important in mediating nociceptive stimuli. However, their precise role in neuropathic pain is still controversial. Here, we have studied the role of epidermal peptidergic nociceptive fibers that are located adjacent to injured fibers in a rat model of neuropathic pain. Using the Spared Nerve Injury (SNI) model, which involves complete transections of the tibial and common peroneal nerve while sparing the sural and saphenous branches, mechanical hypersensitivity was induced of the uninjured lateral (sural) and medial (saphenous) area of the foot sole. At different time points, a complete foot sole biopsy was taken from the injured paw and processed for Calcitonin Gene-Related Peptide (CGRP) immunohistochemistry. Subsequently, a novel 2D-reconstruction model depicting the density of CGRP fibers was made to evaluate the course of denervation and re-innervation by uninjured CGRP fibers. The results show an increased density of uninjured CGRP-IR epidermal fibers on the lateral and medial side after a SNI procedure at 5 and 10 weeks. Furthermore, although in control animals the density of epidermal CGRP-IR fibers in the footpads was lower compared to the surrounding skin of the foot, 10 weeks after the SNI procedure, the initially denervated footpads displayed a hyper-innervation. These data support the idea that uninjured fibers may play a considerable role in development and maintenance of neuropathic pain and that it is important to take larger biopsies to test the relationship between innervation of injured and uninjured nerve areas.

Ins and outs of cerebellar modules.

The modular concept of cerebellar connections has been advocated in the lifetime work of Jan Voogd. In this concept, a cerebellar module is defined as the conglomerate of one or multiple and non-adjacent, parasagittally arranged zones of Purkinje cells, their specific projection to a well-defined region of the cerebellar nuclei, and the climbing fiber input to these zones by a well-defined region of the inferior olivary complex. The modular organization of these olivo-cortico-nuclear connections is further exemplified by matching reciprocal connections between inferior olive and cerebellar nuclei. Because the different regions of the cerebellar nuclei show highly specific output patterns, cerebellar modules have been suggested to constitute functional entities. This idea is strengthened by the observation that anatomically defined modules adhere to the distribution of chemical markers in the cerebellar cortex suggesting that modules not only differ in their input and output relations but also may differ in operational capabilities. Here, I will briefly review some recent data on the establishment of cerebellar modules in rats. Furthermore, some evidence will be shown suggesting that the other main afferent system (i.e., mossy fibers), at least to some extent, also adheres to the modular organization. Finally, using retrograde transneuronal tracing with rabies virus, some evidence will be provided that several cerebellar modules may be involved in the control of individual muscles.

Encoding of whisker input by cerebellar Purkinje cells.

The cerebellar cortex is crucial for sensorimotor integration. Sensorimotor inputs converge on cerebellar Purkinje cells via two afferent pathways: the climbing fibre pathway triggering complex spikes, and the mossy fibre­parallel fibre pathway, modulating the simple spike activities of Purkinje cells. We used, for the first time, the mouse whisker system as a model system to study the encoding of somatosensory input by Purkinje cells.We show that most Purkinje cells in ipsilateral crus 1 and crus 2 of awake mice respond to whisker stimulation with complex spike and/or simple spike responses. Single-whisker stimulation in anaesthetised mice revealed that the receptive fields of complex spike and simple spike responses were strikingly different. Complex spike responses, which proved to be sensitive to the amplitude, speed and direction of whisker movement, were evoked by only one or a few whiskers. Simple spike responses, which were not affected by the direction of movement, could be evoked by many individual whiskers. The receptive fields of Purkinje cells were largely intermingled, and we suggest that this facilitates the rapid integration of sensory inputs from different sources. Furthermore, we describe that individual Purkinje cells, at least under anaesthesia, may be bound in two functional ensembles based on the receptive fields and the synchrony of the complex spike and simple spike responses. The 'complex spike ensembles' were oriented in the sagittal plane, following the anatomical organization of the climbing fibres, while the 'simple spike ensembles' were oriented in the transversal plane, as are the beams of parallel fibres.

Selective impairment of the cerebellar C1 module involved in rat hind limb control reduces step-dependent modulation of cutaneous reflexes.

The cerebellum is divided into multiple parasagittally organized modules, which are thought to represent functional entities. How individual modules participate in cerebellar control of complex movements such as locomotion remains largely unknown. To a large extent, this is caused by the inability to study the contribution of individual modules during locomotion. Because of the architecture of modules, based on narrow, elongated cortical strips that may be discontinuous in the rostrocaudal direction, lesion of a complete module, without affecting neighboring modules, has not been possible. Here, we report on a new method for inducing a selective dysfunction of spatially separated parts of a single module using a small cortical injection of a retrogradely transported neurotoxin, cholera toxin b-subunit-saporin. We show that such a local injection into the C1 module results in climbing fiber and partial mossy fiber deafferentation of functionally related areas of this module, thereby resulting in a severe impairment of the whole module without affecting neighboring modules. A subsequent functional analysis indicates that such an impairment of the hindlimb part of the C1 module did not have a significant impact on skilled walking or overall stepping pattern. However, the modulation of cutaneously induced reflexes during stepping was severely diminished. We propose that the C1 module is specifically involved in the adaptive control of reflexes.

Multiple cerebellar zones are involved in the control of individual muscles: a retrograde transneuronal tracing study with rabies virus in the rat.

To identify cerebellar regions that are involved in the control of limb muscles, rabies virus was injected into the tibialis anterior (TA), the gastrocnemius (GC) or, for comparison, into the flexor digitorum (FD) muscles of the rat. Progression of retrograde transneuronal infection at supraspinal levels was assessed after variable time spans and was divided into three groups. Initially, infected neurons were observed in the reticular formation, lateral vestibular nucleus, red nucleus and motor cortex (group 1). Group 2 was characterized by labelling within the cerebellar nuclei as well as of two vermal strips of Purkinje cells (PCs). Double-labelling with zebrin enabled identification of these strips as the lateral part of the A1- and B-zone. For TA both zones were ipsilateral, whereas for GC the A1 strip predominated contralaterally. Group 3 infections showed additional labelling of multiple, in part bilateral, identifiable strips of PCs in vermis, paravermis and hemisphere. FD injections resulted in less robust labelling of vermal strips and more pronounced labelling within paravermal and hemispheral zonal regions. Only sporadic labelling in corresponding regions of the inferior olive and no labelling of cortical interneurons or granule cells was observed. Prolonged infection was seen to result in degeneration of PCs and possibly of motoneurons. We conclude that vermal, paravermal as well as hemispheral zones of the cerebellar cortex converge upon motoneurons that innervate a particular muscle. In addition, individual zones may control motorpools of different muscles and thus contribute to muscle synergies.

Comparative pathogenesis of rabies in bats and carnivores, and implications for spillover to humans.

Bat-acquired rabies is becoming increasingly common, and its diagnosis could be missed partly because its clinical presentation differs from that of dog-acquired rabies. We reviewed the scientific literature to compare the pathogenesis of rabies in bats and carnivores-including dogs-and related this pathogenesis to differences in the clinical presentation of bat-acquired and dog-acquired rabies in human beings. For bat-acquired rabies, we found that the histological site of exposure is usually limited to the skin, the anatomical site of exposure is more commonly the face, and the virus might be more adapted for entry via the skin than for dog-acquired rabies. These factors could help to explain several differences in clinical presentation between individuals with bat-acquired and those with dog-acquired rabies. A better understanding of these differences should improve the recording of a patient's history, enable drawing up of a more sophisticated list of clinical characteristics, and therefore obtain an earlier diagnosis of rabies after contact with a bat or carnivore that has rabies.

The basal interstitial nucleus (BIN) of the cerebellum provides diffuse ascending inhibitory input to the floccular granule cell layer.

The basal interstitial nucleus (BIN) in the white matter of the vestibulocerebellum has been defined more than three decades ago, but has since been largely ignored. It is still unclear which neurotransmitters are being used by BIN neurons, how these neurons are connected to the rest of the brain and what their activity patterns look like. Here, we studied BIN neurons in a range of mammals, including macaque, human, rat, mouse, rabbit, and ferret, using tracing, immunohistological and electrophysiological approaches. We show that BIN neurons are GABAergic and glycinergic, that in primates they also express the marker for cholinergic neurons choline acetyl transferase (ChAT), that they project with beaded fibers to the glomeruli in the granular layer of the ipsilateral floccular complex, and that they are driven by excitation from the ipsilateral and contralateral medio-dorsal medullary gigantocellular reticular formation. Systematic analysis of codistribution of the inhibitory synapse marker VIAAT, BIN axons, and Golgi cell marker mGluR2 indicate that BIN axon terminals complement Golgi cell axon terminals in glomeruli, accounting for a considerable proportion ( > 20%) of the inhibitory terminals in the granule cell layer of the floccular complex. Together, these data show that BIN neurons represent a novel and relevant inhibitory input to the part of the vestibulocerebellum that controls compensatory and smooth pursuit eye movements.