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.

Specialized connectivity of molecular layer interneuron subtypes leads to disinhibition and synchronous inhibition of cerebellar Purkinje cells.

Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex and are vital to cerebellar processing. MLIs are thought to primarily inhibit Purkinje cells (PCs) and suppress the plasticity of synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs, but the functional significance of these connections is not known. Here, we find that two recently recognized MLI subtypes, MLI1 and MLI2, have a highly specialized connectivity that allows them to serve distinct functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond timescale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent behavior and learning. The synchronous firing of electrically coupled MLI1s and disinhibition provided by MLI2s require a major re-evaluation of cerebellar processing.

Electromyography as a Method for Distinguishing Dystonia in Mice.

Electromyography (EMG) methods allow quantitative analyses of motor function. The techniques include intramuscular recordings that are performed in vivo. However, recording muscle activity in freely moving mice, particularly in models of motor disease, often creates challenges that prevent the acquisition of clean signals. Recording preparations must be stable enough for the experimenter to collect an adequate number of signals for statistical analyses. Instability results in a low signal-to-noise ratio that prohibits proper isolation of EMG signals from the target muscle during the behavior of interest. Such insufficient isolation prevents the analysis of full electrical potential waveforms. In this case, resolving the shape of a waveform to differentiate individual spikes and bursts of muscle activity can be difficult. A common source of instability is an inadequate surgery. Poor surgical techniques cause blood loss, tissue damage, poor healing, encumbered movement, and unstable implantation of the electrodes. Here, we describe an optimized surgical procedure that ensures electrode stability for in vivo muscle recordings. We implement our technique to obtain recordings from agonist and antagonist muscle pairs in the hindlimbs of freely moving adult mice. We validate the stability of our method by holding EMG recordings during dystonic behavior. Our approach is ideal for studying normal and abnormal motor function in actively behaving mice and valuable for recording intramuscular activity when considerable motion is expected.

Deep Brain Stimulation of the Interposed Cerebellar Nuclei in a Conditional Genetic Mouse Model with Dystonia.

Dystonia is a neurological disease that is currently ranked as the third most common motor disorder. Patients exhibit repetitive and sometimes sustained muscle contractions that cause limb and body twisting and abnormal postures that impair movement. Deep brain stimulation (DBS) of the basal ganglia and thalamus can be used to improve motor function when other treatment options fail. Recently, the cerebellum has garnered interest as a DBS target for treating dystonia and other motor disorders. Here, we describe a procedure for targeting DBS electrodes to the interposed cerebellar nuclei to correct motor dysfunction in a mouse model with dystonia. Targeting cerebellar outflow pathways with neuromodulation opens new possibilities for using the expansive connectivity of the cerebellum to treat motor and non-motor diseases.

Cerebellar circuits for disinhibition and synchronous inhibition.

The cerebellar cortex contributes to diverse behaviors by transforming mossy fiber inputs into predictions in the form of Purkinje cell (PC) outputs, and then refining those predictions1. Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex2, and are vital to cerebellar processing1,3. MLIs are thought to primarily inhibit PCs and suppress the plasticity of excitatory synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs4-7, but the functional significance of these connections is not known1,3. Behavioral studies suggest that cerebellar-dependent learning is gated by disinhibition of PCs, but the source of such disinhibition has not been identified8. Here we find that two recently recognized MLI subtypes2, MLI1 and MLI2, have highly specialized connectivity that allows them to serve very different functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond time scale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, they primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent learning8. These findings require a major reevaluation of processing within the cerebellum in which disinhibition, a powerful circuit motif present in the cerebral cortex and elsewhere9-17, greatly increases the computational power and flexibility of the cerebellum. They also suggest that millisecond time scale synchronous firing of electrically-coupled MLI1s helps regulate the output of the cerebellar cortex by synchronously pausing PC firing, which has been shown to evoke precisely-timed firing in PC targets18.

Maturation of Purkinje cell firing properties relies on neurogenesis of excitatory neurons.

Preterm infants that suffer cerebellar insults often develop motor disorders and cognitive difficulty. Excitatory granule cells, the most numerous neuron type in the brain, are especially vulnerable and likely instigate disease by impairing the function of their targets, the Purkinje cells. Here, we use regional genetic manipulations and in vivo electrophysiology to test whether excitatory neurons establish the firing properties of Purkinje cells during postnatal mouse development. We generated mutant mice that lack the majority of excitatory cerebellar neurons and tracked the structural and functional consequences on Purkinje cells. We reveal that Purkinje cells fail to acquire their typical morphology and connectivity, and that the concomitant transformation of Purkinje cell firing activity does not occur either. We also show that our mutant pups have impaired motor behaviors and vocal skills. These data argue that excitatory cerebellar neurons define the maturation time-window for postnatal Purkinje cell functions and refine cerebellar-dependent behaviors.

Preterm infants have a higher risk of developing movement difficulties and neurodevelopmental conditions like autism spectrum disorder. This is likely caused by injuries to a part of the brain called the cerebellum. The cerebellum is important for movement, language and social interactions. During the final weeks of pregnancy, the cerebellum grows larger and develops a complex pattern of folds. Tiny granule cells, which are particularly vulnerable to harm, drive this development. Exactly how damage to granule cells causes movement difficulties and other conditions is unclear. One potential explanation may be that granule cells are important for the development of Purkinje cells in the brain. The Purkinje cells send and receive messages and are very important for coordinating movement. To learn more, van der Heijden et al. studied Purkinje cells in mice during a period that corresponds with the third trimester of pregnancy in humans. During this time, the pattern of electrical signals sent by the Purkinje cells changed from slow and irregular to fast and rhythmic with long pauses between bursts. However, mice that had been genetically engineered to lack most of their granule cells showed a completely different pattern of Purkinje cell development. The pattern of electrical signals emitted by these Purkinje cells stayed slow and irregular. Mice that lacked granule cells also had movement difficulties, tremors, and abnormal vocalizations. The experiments confirm that granule cells are essential for normal brain development. Without enough granule cells, the Purkinje cells become stuck in an immature state. This discovery may help physicians identify preterm infants with motor disorders and other conditions earlier. It may also lead to changes in the care of preterm infants designed to protect their granule cells.

Eph/ephrin Function Contributes to the Patterning of Spinocerebellar Mossy Fibers Into Parasagittal Zones.

Purkinje cell microcircuits perform diverse functions using widespread inputs from the brain and spinal cord. The formation of these functional circuits depends on developmental programs and molecular pathways that organize mossy fiber afferents from different sources into a complex and precisely patterned map within the granular layer of the cerebellum. During development, Purkinje cell zonal patterns are thought to guide mossy fiber terminals into zones. However, the molecular mechanisms that mediate this process remain unclear. Here, we used knockout mice to test whether Eph/ephrin signaling controls Purkinje cell-mossy fiber interactions during cerebellar circuit formation. Loss of ephrin-A2 and ephrin-A5 disrupted the patterning of spinocerebellar terminals into discrete zones. Zone territories in the granular layer that normally have limited spinocerebellar input contained ectopic terminals in ephrin-A2 -/-;ephrin-A5 -/- double knockout mice. However, the overall morphology of the cerebellum, lobule position, and Purkinje cell zonal patterns developed normally in the ephrin-A2 -/-;ephrin-A5 -/- mutant mice. This work suggests that communication between Purkinje cell zones and mossy fibers during postnatal development allows contact-dependent molecular cues to sharpen the innervation of sensory afferents into functional zones.

Purkinje cell neurotransmission patterns cerebellar basket cells into zonal modules defined by distinct pinceau sizes.

Ramón y Cajal proclaimed the neuron doctrine based on circuit features he exemplified using cerebellar basket cell projections. Basket cells form dense inhibitory plexuses that wrap Purkinje cell somata and terminate as pinceaux at the initial segment of axons. Here, we demonstrate that HCN1, Kv1.1, PSD95 and GAD67 unexpectedly mark patterns of basket cell pinceaux that map onto Purkinje cell functional zones. Using cell-specific genetic tracing with an Ascl1CreERT2 mouse conditional allele, we reveal that basket cell zones comprise different sizes of pinceaux. We tested whether Purkinje cells instruct the assembly of inhibitory projections into zones, as they do for excitatory afferents. Genetically silencing Purkinje cell neurotransmission blocks the formation of sharp Purkinje cell zones and disrupts excitatory axon patterning. The distribution of pinceaux into size-specific zones is eliminated without Purkinje cell GABAergic output. Our data uncover the cellular and molecular diversity of a foundational synapse that revolutionized neuroscience.

Bifidobacteria shape host neural circuits during postnatal development by promoting synapse formation and microglial function.

We hypothesized that early-life gut microbiota support the functional organization of neural circuitry in the brain via regulation of synaptic gene expression and modulation of microglial functionality. Germ-free mice were colonized as neonates with either a simplified human infant microbiota consortium consisting of four Bifidobacterium species, or with a complex, conventional murine microbiota. We examined the cerebellum, cortex, and hippocampus of both groups of colonized mice in addition to germ-free control mice. At postnatal day 4 (P4), conventionalized mice and Bifidobacterium-colonized mice exhibited decreased expression of synapse-promoting genes and increased markers indicative of reactive microglia in the cerebellum, cortex and hippocampus relative to germ-free mice. By P20, both conventional and Bifidobacterium-treated mice exhibited normal synaptic density and neuronal activity as measured by density of VGLUT2+ puncta and Purkinje cell firing rate respectively, in contrast to the increased synaptic density and decreased firing rate observed in germ-free mice. The conclusions from this study further reveal how bifidobacteria participate in establishing functional neural circuits. Collectively, these data indicate that neonatal microbial colonization of the gut elicits concomitant effects on the host CNS, which promote the homeostatic developmental balance of neural connections during the postnatal time period.

Molecular layer interneurons shape the spike activity of cerebellar Purkinje cells.

Purkinje cells receive synaptic input from several classes of interneurons. Here, we address the roles of inhibitory molecular layer interneurons in establishing Purkinje cell function in vivo. Using conditional genetics approaches in mice, we compare how the lack of stellate cell versus basket cell GABAergic neurotransmission sculpts the firing properties of Purkinje cells. We take advantage of an inducible Ascl1CreER allele to spatially and temporally target the deletion of the vesicular GABA transporter, Vgat, in developing neurons. Selective depletion of basket cell GABAergic neurotransmission increases the frequency of Purkinje cell simple spike firing and decreases the frequency of complex spike firing in adult behaving mice. In contrast, lack of stellate cell communication increases the regularity of Purkinje cell simple spike firing while increasing the frequency of complex spike firing. Our data uncover complementary roles for molecular layer interneurons in shaping the rate and pattern of Purkinje cell activity in vivo.

Recent advances in understanding the mechanisms of cerebellar granule cell development and function and their contribution to behavior.

The cerebellum is the focus of an emergent series of debates because its circuitry is now thought to encode an unexpected level of functional diversity. The flexibility that is built into the cerebellar circuit allows it to participate not only in motor behaviors involving coordination, learning, and balance but also in non-motor behaviors such as cognition, emotion, and spatial navigation. In accordance with the cerebellum's diverse functional roles, when these circuits are altered because of disease or injury, the behavioral outcomes range from neurological conditions such as ataxia, dystonia, and tremor to neuropsychiatric conditions, including autism spectrum disorders, schizophrenia, and attention-deficit/hyperactivity disorder. Two major questions arise: what types of cells mediate these normal and abnormal processes, and how might they accomplish these seemingly disparate functions? The tiny but numerous cerebellar granule cells may hold answers to these questions. Here, we discuss recent advances in understanding how the granule cell lineage arises in the embryo and how a stem cell niche that replenishes granule cells influences wiring when the postnatal cerebellum is injured. We discuss how precisely coordinated developmental programs, gene expression patterns, and epigenetic mechanisms determine the formation of synapses that integrate multi-modal inputs onto single granule cells. These data lead us to consider how granule cell synaptic heterogeneity promotes sensorimotor and non-sensorimotor signals in behaving animals. We discuss evidence that granule cells use ultrafast neurotransmission that can operate at kilohertz frequencies. Together, these data inspire an emerging view for how granule cells contribute to the shaping of complex animal behaviors.

Shaping Diversity Into the Brain's Form and Function.

The brain contains a large diversity of unique cell types that use specific genetic programs to control development and instruct the intricate wiring of sensory, motor, and cognitive brain regions. In addition to their cellular diversity and specialized connectivity maps, each region's dedicated function is also expressed in their characteristic gross external morphologies. The folds on the surface of the cerebral cortex and cerebellum are classic examples. But, to what extent does structure relate to function and at what spatial scale? We discuss the mechanisms that sculpt functional brain maps and external morphologies. We also contrast the cryptic structural defects in conditions such as autism spectrum disorders to the overt microcephaly after Zika infections, taking into consideration that both diseases disrupt proper cognitive development. The data indicate that dynamic processes shape all brain areas to fit into jigsaw-like patterns. The patterns in each region reflect circuit connectivity, which ultimately supports local signal processing and accomplishes multi-areal integration of information processing to optimize brain functions.

ATXN1-CIC Complex Is the Primary Driver of Cerebellar Pathology in Spinocerebellar Ataxia Type 1 through a Gain-of-Function Mechanism.

Polyglutamine (polyQ) diseases are caused by expansion of translated CAG repeats in distinct genes leading to altered protein function. In spinocerebellar ataxia type 1 (SCA1), a gain of function of polyQ-expanded ataxin-1 (ATXN1) contributes to cerebellar pathology. The extent to which cerebellar toxicity depends on its cognate partner capicua (CIC), versus other interactors, remains unclear. It is also not established whether loss of the ATXN1-CIC complex in the cerebellum contributes to disease pathogenesis. In this study, we exclusively disrupt the ATXN1-CIC interaction in vivo and show that it is at the crux of cerebellar toxicity in SCA1. Importantly, loss of CIC in the cerebellum does not cause ataxia or Purkinje cell degeneration. Expression profiling of these gain- and loss-of-function models, coupled with data from iPSC-derived neurons from SCA1 patients, supports a mechanism in which gain of function of the ATXN1-CIC complex is the major driver of toxicity.

WGA-Alexa Conjugates for Axonal Tracing.

Anatomical labeling approaches are essential for understanding brain organization. Among these approaches are various methods of performing tract tracing. However, a major hurdle to overcome when marking neurons in vivo is visibility. Poor visibility makes it challenging to image a desired neuronal pathway so that it can be easily differentiated from a closely neighboring pathway. As a result, it becomes impossible to analyze individual projections or their connections. The tracer that is chosen for a given purpose has a major influence on the quality of the tracing. Here, we describe the wheat germ agglutinin (WGA) tracer conjugated to Alexa fluorophores for reliable high-resolution tracing of central nervous system projections. Using the mouse cerebellum as a model system, we implement WGA-Alexa tracing for marking and mapping neural circuits that control motor function. We also show its utility for marking localized regions of the cerebellum after performing single-unit extracellular recordings in vivo. © 2017 by John Wiley & Sons, Inc.

An optimized surgical approach for obtaining stable extracellular single-unit recordings from the cerebellum of head-fixed behaving mice.

BACKGROUND: Electrophysiological recording approaches are essential for understanding brain function. Among these approaches are various methods of performing single-unit recordings. However, a major hurdle to overcome when recording single units in vivo is stability. Poor stability results in a low signal-to-noise ratio, which makes it challenging to isolate neuronal signals. Proper isolation is needed for differentiating a signal from neighboring cells or the noise inherent to electrophysiology. Insufficient isolation makes it impossible to analyze full action potential waveforms. A common source of instability is an inadequate surgery. Problems during surgery cause blood loss, tissue damage and poor healing of the surrounding tissue, limited access to the target brain region, and, importantly, unreliable fixation points for holding the mouse's head. NEW METHOD: We describe an optimized surgical procedure that ensures limited tissue damage and delineate a method for implanting head plates to hold the animal firmly in place. RESULTS: Using the cerebellum as a model, we implement an extracellular recording technique to acquire single units from Purkinje cells and cerebellar nuclear neurons in behaving mice. We validate the stability of our method by holding single units after injecting the powerful tremorgenic drug harmaline. We performed multiple structural analyses after recording. COMPARISON WITH EXISTING METHODS: Our approach is ideal for studying neuronal function in active mice and valuable for recording single-neuron activity when considerable motion is unavoidable. CONCLUSIONS: The surgical principles we present for accessing the cerebellum can be easily adapted to examine the function of neurons in other brain regions.

Development of myelination and cholinergic innervation in the central auditory system of a prosimian primate (Otolemur garnetti).

Change in the timeline of neurobiological growth is an important source of biological variation, and thus phenotypic evolution. However, no study has to date investigated sensory system development in any of the prosimian primates that are thought to most closely resemble our earliest primate ancestors. Acetylcholine (ACh) is a neurotransmitter critical to normal brain function by regulating synaptic plasticity associated with attention and learning. Myelination is an important structural component of the brain because it facilitates rapid neuronal communication. In this work we investigated the expression of acetylcholinesterase (AChE) and the density of myelinated axons throughout postnatal development in the inferior colliculus (IC), medial geniculate complex (MGC), and auditory cortex (auditory core, belt, and parabelt) in Garnett's greater galago (Otolemur garnetti). We found that the IC and MGC exhibit relatively high myelinated fiber length density (MFLD) values at birth and attain adult-like values by the species-typical age at weaning. In contrast, neocortical auditory fields are relatively unmyelinated at birth and only attain adult-like MFLD values by the species-typical age at puberty. Analysis of AChE expression indicated that, in contrast to evidence from rodent samples, the adult-like distribution of AChE in the core area of auditory cortex, dense bands in layers I, IIIb/IV, and Vb/VI, is present at birth. These data indicate the differential developmental trajectory of central auditory system structures and demonstrate the early onset of adult-like AChE expression in primary auditory cortex in O. garnetti, suggesting the auditory system is more developed at birth in primates compared to rodents.