Inferior Olivary Neurons Research Articles

Two-Photon Laser Ablation and In Vivo Wide-Field Imaging of Inferior Olive Neurons Revealed the Recovery of Olivocerebellar Circuits in Zebrafish.

The cerebellum, a brain region with a high degree of plasticity, is pivotal in motor control, learning, and cognition. The cerebellar reserve is the capacity of the cerebellum to respond and adapt to various disorders via resilience and reversibility. Although structural and functional recovery has been reported in mammals and has attracted attention regarding treatments for cerebellar dysfunction, such as spinocerebellar degeneration, the regulatory mechanisms of the cerebellar reserve are largely unidentified, particularly at the circuit level. Herein, we established an optical approach using zebrafish, an ideal vertebrate model in optical techniques, neuroscience, and developmental biology. By combining two-photon laser ablation of the inferior olive (IO) and long-term non-invasive imaging of “the whole brain” at a single-cell resolution, we succeeded in visualization of the morphological changes occurring in the IO neuron population and showed at a single-cell level that structural remodeling of the olivocerebellar circuit occurred in a relatively short period. This system, in combination with various functional analyses, represents a novel and powerful approach for uncovering the mechanisms of the cerebellar reserve, and highlights the potential of the zebrafish model to elucidate the organizing principles of neuronal circuits and their homeostasis in health and disease.

<em>In vivo</em> 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.

Influence of various temporal recoding on pavlovian eyeblink conditioning in the cerebellum

We consider the Pavlovian eyeblink conditioning (EBC) via repeated presentation of paired conditioned stimulus (tone) and unconditioned stimulus (US; airpuff). In an effective cerebellar ring network, we change the connection probability from Golgi to granule (GR) cells, and make a dynamical classification of various firing patterns of the GR cells. Individual GR cells are thus found to show various well- and ill-matched firing patterns relative to the US timing signal. Then, these variously-recoded signals are fed into the Purkinje cells (PCs) through the parallel-fibers (PFs). Based on such unique dynamical classification of various firing patterns, we make intensive investigations on the influence of various temporal recoding (i.e., firing patterns) of the GR cells on the synaptic plasticity of the PF-PC synapses and the subsequent learning process for the EBC. We first note that the variously-recoded PF signals are effectively depressed by the (error-teaching) instructor climbing-fiber (CF) signals from the inferior olive neuron. In the case of well-matched PF signals, they are strongly depressed through strong long-term depression (LTD) by the instructor CF signals due to good association between the in-phase PF and the instructor CF signals. On the other hand, practically no LTD occurs for the ill-matched PF signals because most of them have no association with the instructor CF signals. This kind of "effective" depression at the PF-PC synapses coordinates firings of PCs effectively, which then makes effective inhibitory coordination on the cerebellar nucleus neuron [which elicits conditioned response (CR; eyeblink)]. When the learning trial passes a threshold, acquisition of CR begins. In this case, the timing degree of CR becomes good due to presence of the ill-matched firing group which plays a role of protection barrier for the timing. With further increase in the number of trials, strength of CR (corresponding to the amplitude of eyelid closure) increases due to strong LTD in the well-matched firing group, while its timing degree decreases. In this way, the well- and the ill-matched firing groups play their own roles for the strength and the timing of CR, respectively. Thus, with increasing the number of learning trials, the (overall) learning efficiency degree (taking into consideration both timing and strength of CR) for the CR is increased, and eventually it becomes saturated. Finally, we also discuss dependence of the variety degree for firing patterns of the GR cells and the saturated learning efficiency degree of the CR on and their relations.

Anoctamin 2-chloride channels reduce simple spike activity and mediate inhibition at elevated calcium concentration in cerebellar Purkinje cells.

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning.

Kv11 (ether-à-go-go-related gene) voltage-dependent K+ channels promote resonance and oscillation of subthreshold membrane potentials.

Key points Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so‐called ‘resonant properties’).Here, we report that heterologous expression of mouse Kv11 voltage‐dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties.Kv11.3‐expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor.The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice.This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor‐like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+) channels, encoded by ether‐á‐go‐go‐related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole‐cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3‐, Kv11.1(Merg1b)‐ and Kv11.1(Merg1a)‐expressing cells were 2–6 Hz, 2–4 Hz, and 0.6–0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor–inductor–capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11‐dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at –30 to –60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency‐dependent responses in a subtype‐dependent manner from resting to depolarized membrane potentials.

Cyclin-dependent kinase 5/p35 contributes synergistically with Reelin/Dab1 to the positioning of facial branchiomotor and inferior olive neurons in the developing mouse hindbrain.

Cyclin-dependent kinase 5 (Cdk5)/p35 is a serine/threonine kinase, and its activity is detected primarily in postmitotic neurons. Mice lacking Cdk5/p35 display migration defects of the cortical neurons in the cerebrum and cerebellum. In this study, we demonstrate that although most brainstem nuclei are found in their proper positions, the motor nucleus of the facial nerve is ectopically located and neurons of the inferior olive fail to position correctly, resulting in the lack of their characteristic structures in the hindbrain of Cdk5-/- mice. Despite the defective migration of these neurons, axonal exits of the facial nerve from brainstem and projections of the inferior cerebellar axons appear unchanged in Cdk5-/- mice. Defective neuronal migration in Cdk5-/- hindbrain was rescued by the neuron-specific expression of Cdk5 transgene. Because developmental defects of these structures have been reported in reeler and Dab1 mutant mice, we analyzed the double-null mutants of p35 and Dab1 and found more extensive ectopia of VII motor nuclei in these mice. These results indicate that Cdk5/p35 and Reelin signaling regulates the selective mode of neuronal migration in the developing mouse hindbrain.

Increased Purkinje Cell Complex Spike and Deep Cerebellar Nucleus Synchrony as a Potential Basis for Syndromic Essential Tremor. A Review and Synthesis of the Literature.

We review advances in understanding Purkinje cell (PC) complex spike (CS) physiology that suggest increased CS synchrony underlies syndromic essential tremor (ET). We searched PubMed for papers describing factors that affect CS synchrony or cerebellar circuits potentially related to tremor. Inferior olivary (IO) neurons are electrically coupled, with the degree of coupling controlled by excitatory and GABAergic inputs. Clusters of coupled IO neurons synchronize CSs within parasagittal bands via climbing fibers (Cfs). When motor cortex is stimulated in rats at varying frequencies, whisker movement occurs at ~10Hz, correlated with synchronous CSs, indicating that the IO/CS oscillatory rhythm gates movement frequency. Intra-IO injection of the GABAA receptor antagonist picrotoxin increases CS synchrony, increases whisker movement amplitude, and induces tremor. Harmaline and 5-HT2a receptor activation also increase IO coupling and CS synchrony and induce tremor. The hotfoot17 mouse displays features found in ET brains, including cerebellar GluRδ2 deficiency and abnormal PC Cf innervation, with IO- and PC-dependent cerebellar oscillations and tremor likely due to enhanced CS synchrony. Heightened coupling within the IO oscillator leads, through its dynamic control of CS synchrony, to increased movement amplitude and, when sufficiently intense, action tremor. Increased CS synchrony secondary to aberrant Cf innervation of multiple PCs likely also underlies hotfoot17 tremor. Deep cerebellar nucleus (DCN) hypersynchrony may occur secondary to increased CS synchrony but might also occur from PC axonal terminal sprouting during partial PC loss. Through these combined mechanisms, increased CS/DCN synchrony may plausibly underlie syndromic ET.

Climbing fiber synapses rapidly and transiently inhibit neighboring Purkinje cells via ephaptic coupling

Climbing fibers (CFs) from the inferior olive (IO) make strong excitatory synapses onto cerebellar Purkinje cell (PC) dendrites, and trigger distinctive responses known as complex spikes (CSs). We find that in awake mice, a CS in one PC suppresses conventional simple spikes (SSs) in neighboring PCs for several milliseconds. This involves a novel ephaptic coupling, in which an excitatory synapse generates large negative extracellular signals that nonsynaptically inhibit neighboring PCs. The distance dependence of CS-SS ephaptic signaling, combined with the known CF divergence, allows a single IO neuron to influence the output of the cerebellum by synchronously suppressing the firing of potentially over one hundred PCs. Optogenetic studies in vivo, and dynamic clamp studies in slice, indicate that such brief PC suppression, either as a result of ephaptic signaling or other mechanisms, can effectively promote firing in neurons in the deep cerebellar nuclei with remarkable speed and precision.

Electrical coupling controls dimensionality and chaotic firing of inferior olive neurons.

We previously proposed, on theoretical grounds, that the cerebellum must regulate the dimensionality of its neuronal activity during motor learning and control to cope with the low firing frequency of inferior olive neurons, which form one of two major inputs to the cerebellar cortex. Such dimensionality regulation is possible via modulation of electrical coupling through the gap junctions between inferior olive neurons by inhibitory GABAergic synapses. In addition, we previously showed in simulations that intermediate coupling strengths induce chaotic firing of inferior olive neurons and increase their information carrying capacity. However, there is no in vivo experimental data supporting these two theoretical predictions. Here, we computed the levels of synchrony, dimensionality, and chaos of the inferior olive code by analyzing in vivo recordings of Purkinje cell complex spike activity in three different coupling conditions: carbenoxolone (gap junctions blocker), control, and picrotoxin (GABA-A receptor antagonist). To examine the effect of electrical coupling on dimensionality and chaotic dynamics, we first determined the physiological range of effective coupling strengths between inferior olive neurons in the three conditions using a combination of a biophysical network model of the inferior olive and a novel Bayesian model averaging approach. We found that effective coupling co-varied with synchrony and was inversely related to the dimensionality of inferior olive firing dynamics, as measured via a principal component analysis of the spike trains in each condition. Furthermore, for both the model and the data, we found an inverted U-shaped relationship between coupling strengths and complexity entropy, a measure of chaos for spiking neural data. These results are consistent with our hypothesis according to which electrical coupling regulates the dimensionality and the complexity in the inferior olive neurons in order to optimize both motor learning and control of high dimensional motor systems by the cerebellum.

Modular output circuits of the fastigial nucleus for diverse motor and nonmotor functions of the cerebellar vermis.

The cerebellar vermis, long associated with axial motor control, has been implicated in a surprising range of neuropsychiatric disorders and cognitive and affective functions. Remarkably little is known, however, about the specific cell types and neural circuits responsible for these diverse functions. Here, using single-cell gene expression profiling and anatomical circuit analyses of vermis output neurons in the mouse fastigial (medial cerebellar) nucleus, we identify five major classes of glutamatergic projection neurons distinguished by gene expression, morphology, distribution, and input-output connectivity. Each fastigial cell type is connected with a specific set of Purkinje cells and inferior olive neurons and in turn innervates a distinct collection of downstream targets. Transsynaptic tracing indicates extensive disynaptic links with cognitive, affective, and motor forebrain circuits. These results indicate that diverse cerebellar vermis functions could be mediated by modular synaptic connections of distinct fastigial cell types with posturomotor, oromotor, positional-autonomic, orienting, and vigilance circuits.

Selective neuronal vulnerability is involved in cerebellar lesions of Guinea pigs infected with bovine spongiform encephalopathy (BSE) prions: Immunohistochemical and electron microscopic investigations.

The cerebellar lesions of bovine spongiform encephalopathy (BSE)-infected guinea pigs were characterized as severe atrophy of the cerebellar cortex associated with the loss of granule cells, decrease in the width of the molecular layer, and intense protease-resistant prion protein (PrPSc ) accumulations that are similar to cerebellar lesions in kuru and the VV2 type of sporadic Creutzfeldt-Jakob disease. The aim of this study is to assess the relationships between the distribution and localization of PrPSc and synapses expressing neurotransmitter transporters in order to reveal the pathogenesis of the disease. We used cell-type-specific immunohistochemical makers recognizing glutamatergic and γ-aminobutylic acid (GABA)ergic terminals to identify terminals impaired with PrPSc accumulations. The distribution of PrPSc accumulations and immunoreactivity of synaptic vesicles were studied throughout the neuroanatomical pathways in cerebellar lesions. Time course study demonstrated that PrPSc accumulation showed a tendency to spread from granular layer to molecular layer. The immunoreactivity of vesicular glutamate transporter 1 (VGluT1) was localized in axon terminals of cerebellar granule cells, and decreased in association with the severity of PrPSc accumulations and loss of granule cells. Immunoreactivities of vesicular glutamate transporter 2 (VGluT2) and vesicular GABA transporter (VGAT) that exist in axon terminals of inferior olivary neurons and GABAergic synapses of Purkinje cells, respectively, were preserved well in these lesions. In brainstem, VGluT1 immunoreactivity decreased selectively in pontine nuclei that are a component of the pontocerebellar pathway, although other neurotransmitter immunoreactivities were preserved well. Our findings suggest that the selective loss of VGluT1-immunoreactive synapses subsequent to PrPSc accumulations can contribute to the pathogenesis of cerebellar lesions of BSE-infected guinea pigs.

Functional Antagonism of Sphingosine-1-Phosphate Receptor 1 Prevents Harmaline-Induced Ultrastructural Alterations and Caspase-3 Mediated Apoptosis.

BackgroundThere is a meaningful necessity for a targeted therapy of essential tremor (ET), as medications have not been developed specifically for ET. For nearly a century, many drugs have been applied in the treatment of tremor but the drug treatment of ET remains still unknown. Some potential therapeutic factors such fingolimod (FTY720) can be effectively used to treat ET in animals. In the present research, the effect of FTY720, the immunomodulatory sphingosine 1-phosphate (S1P) analog, on degeneration of cerebellar and olivary neurons induced by harmaline in male rats was investigated.MethodsThe animals were allotted into control dimethyl sulfoxide (DMSO), saline + harmaline [30 mg/kg, intraperitoneally, (i.p.)], harmaline + FTY720 (1 mg/kg, i.p, 1 h and 24 h before harmaline injection) groups (n = 10). The cerebellum and inferior olive nucleus (ION) were studied for neuronal degeneration using immunohistochemistry (IHC) and ultrastructural study by transmission electron microscopy (TEM) techniques.ResultsHarmaline caused neuronal cell loss, caspase-3 mediated apoptosis, astrocytosis and ultrastructural changes in cerebellar Purkinje cells and inferior olive neurons. FTY720 exhibited neuroprotective effects on cerebellar Purkinje cells and inferior olivary neurons.ConclusionThese results suggest that FTY720 has potential efficacy for prevention of ET neurodegeneration and astrocytosis induced by harmaline in male rats.

Electrical Coupling Controls Dimensionality and Chaotic Firing of Inferior Olive Neurons

One of the main challenges facing online neural learning systems with numerous modifiable parameters (or “degrees-of-freedom”) such as the cerebellum, is how to avoid “overfitting”. We previously proposed that the cerebellum controls the degree-of-freedoms during learning by gradually modulating the electric coupling strength between inferior olive neurons. Here, we develop a modeling technique to estimate effective coupling strengths between inferior olive neurons from in vivo recordings of Purkinje cell complex spike activity in three different coupling conditions. We show that high coupling strengths induce synchronous firing and decrease the dimensionality of inferior olive firing dynamics. In contrast, intermediate coupling strengths induce chaotic firing and increase the dimensionality of firing dynamics. Our results thus support the hypothesis that effective coupling controls the dimensionality of inferior olive firing, which may allow the olivocerebellar system to learn effectively from a small training sample set despite the low firing frequency of inferior olive neurons.

Neurons of the inferior olive respond to broad classes of sensory input while subject to homeostatic control.

Key points Purkinje cells in the cerebellum integrate input from sensory organs with that from premotor centres.Purkinje cells use a variety of sensory inputs relaying information from the environment to modify motor control.Here we investigated to what extent the climbing fibre inputs to Purkinje cells signal mono‐ or multi‐sensory information, and to what extent this signalling is subject to recent history of activity.We show that individual climbing fibres convey multiple types of sensory information, together providing a rich mosaic projection pattern of sensory signals across the cerebellar cortex.Moreover, firing probability of climbing fibres following sensory stimulation depends strongly on the recent history of activity, showing a tendency to homeostatic dampening. Cerebellar Purkinje cells integrate sensory information with motor efference copies to adapt movements to behavioural and environmental requirements. They produce complex spikes that are triggered by the activity of climbing fibres originating in neurons of the inferior olive. These complex spikes can shape the onset, amplitude and direction of movements and the adaptation of such movements to sensory feedback. Clusters of nearby inferior olive neurons project to parasagittally aligned stripes of Purkinje cells, referred to as ‘microzones’. It is currently unclear to what extent individual Purkinje cells within a single microzone integrate climbing fibre inputs from multiple sources of different sensory origins, and to what extent sensory‐evoked climbing fibre responses depend on the strength and recent history of activation. Here we imaged complex spike responses in cerebellar lobule crus 1 to various types of sensory stimulation in awake mice. We find that different sensory modalities and receptive fields have a mild, but consistent, tendency to converge on individual Purkinje cells, with climbing fibres showing some degree of input‐specificity. Purkinje cells encoding the same stimulus show increased events with coherent complex spike firing and tend to lie close together. Moreover, whereas complex spike firing is only mildly affected by variations in stimulus strength, it depends strongly on the recent history of climbing fibre activity. Our data point towards a mechanism in the olivo‐cerebellar system that regulates complex spike firing during mono‐ or multi‐sensory stimulation around a relatively low set‐point, highlighting an integrative coding scheme of complex spike firing under homeostatic control.

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.

Mechanism Underlying Organophosphate Paraoxon-Induced Kinetic Tremor.

Organophosphates (OPs) inhibit cholinesterase and hyperactivate the acetylcholinergic nervous system in the brain, causing motor disorders (e.g., tremor and seizures). Here, we performed behavioral and immunohistochemical studies in mice and rats to investigate the tremorgenic mechanism of paraoxon, an active metabolite of parathion. Treating animals with paraoxon (0.15-0.6mg/kg, i.p.) elicited kinetic tremor in a dose-dependent manner. Expressional analysis of Fos protein, a biomarker of neural excitation, revealed that a tremorgenic dose of paraoxon (0.6mg/kg) significantly and region-specifically elevated Fos expression in the cerebral cortex (e.g., sensory cortex), hippocampal CA1, globus pallidus, medial habenula, and inferior olive (IO) among 48 brain regions examined. A moderate increase in Fos expression was also observed in the dorsolateral striatum while the change was not statistically significant. Paraoxon-induced tremor was inhibited by the nicotinic acetylcholine (nACh) receptor antagonist mecamylamine (MEC), but not affected by the muscarinic acetylcholine receptor antagonist trihexyphenidyl (THP). In addition, paraoxon-induced Fos expression in the IO was also antagonized by MEC, but not by THP, and lesioning of the IO markedly suppressed tremorgenic action of paraoxon. The present results suggest that OPs elicit kinetic tremor at least partly by activating IO neurons via nACh receptors.

Corollary discharge in precerebellar nuclei of sleeping infant rats.

In week-old rats, somatosensory input arises predominantly from external stimuli or from sensory feedback (reafference) associated with myoclonic twitches during active sleep. A previous study suggested that the brainstem motor structures that produce twitches also send motor copies (or corollary discharge, CD) to the cerebellum. We tested this possibility by recording from two precerebellar nuclei-the inferior olive (IO) and lateral reticular nucleus (LRN). In most IO and LRN neurons, twitch-related activity peaked sharply around twitch onset, consistent with CD. Next, we identified twitch-production areas in the midbrain that project independently to the IO and LRN. Finally, we blocked calcium-activated slow potassium (SK) channels in the IO to explain how broadly tuned brainstem motor signals can be transformed into precise CD signals. We conclude that the precerebellar nuclei convey a diversity of sleep-related neural activity to the developing cerebellum to enable processing of convergent input from CD and reafferent signals.

Multiple Phases of Climbing Fiber Synapse Elimination in the Developing Cerebellum.

Functional neural circuits in the mature animals are shaped during postnatal development by elimination of unnecessary synapses and strengthening of necessary ones among redundant synaptic connections formed transiently around birth. In the cerebellum of neonatal rodents, excitatory synapses are formed on the somata of Purkinje cells (PCs) by climbing fibers (CFs) that originate from neurons in the contralateral inferior olive. Each PC receives inputs from multiple (~ five) CFs that have about equal synaptic strengths. Subsequently, a single CF selectively becomes stronger relative to the other CFs during the first postnatal week. Then, from around postnatal day 9 (P9), only the strongest CF ("winner" CF) extends its synaptic territory along PC dendrites. In contrast, synapses of the weaker CFs ("loser" CFs) remain on the soma and the most proximal portion of the dendrite together with somatic synapses of the "winner" CF. These perisomatic CF synapses are eliminated progressively during the second and the third postnatal weeks. From P6 to P11, the elimination proceeds independently of the formation of the synapses on PC dendrites by parallel fibers (PFs). From P12 and thereafter, the elimination requires normal PF-PC synapse formation and is presumably dependent on the PF synaptic inputs. Most PCs become mono-innervated by single strong CFs on their dendrites in the third postnatal week. In this review article, we will describe how adult-type CF mono-innervation of PC is established through these multiple phases of postnatal cerebellar development and make an overview of molecular/cellular mechanisms underlying them.

In Vivo Analysis of the Climbing Fiber-Purkinje Cell Circuit in SCA2-58Q Transgenic Mouse Model.

Cerebellar Purkinje cells (PCs) and cerebellar pathways are primarily affected in many autosomal dominant cerebellar ataxias. PCs generate complex spikes (CS) in vivo when activated by climbing fiber (CF) which rise from the inferior olive. In this study, we investigated the functional state of the CF-PC circuitry in the transgenic mouse model of spinocerebellar ataxia type 2 (SCA2), a polyglutamine neurodegenerative genetic disease. In our experiments, we used an extracellular single-unit recording method to compare the PC activity pattern and the CS shape in age-matched wild-type mice and SCA2-58Q transgenic mice. We discovered no alterations in the CS properties of PCs in aging SCA2 mice. To examine the integrity of the olivocerebellar pathway, we applied harmaline, an alkaloid that acts directly on the inferior olive neurons. The pharmacological stimulation of olivocerebellar circuit by harmaline uncovered disturbances in SCA2-58Q PC activity pattern and in the complex spike shape when compared with age-matched wild-type cells. The abnormalities in the CF-PC circuitry were aggravated with age. We propose that alterations in CF-PC circuitry represent one of potential causes of ataxic symptoms in SCA2 and in other SCAs.

Inferior Olivary TMEM16B Mediates Cerebellar Motor Learning

Ca2+-activated ion channels shape membrane excitability and Ca2+ dynamics in response to cytoplasmic Ca2+ elevation. Compared to the Ca2+-activated K+ channels, known as BK and SK channels, the physiological importance of Ca2+-activated Cl− channels (CaCCs) in neurons has been largely overlooked. Here we report that CaCCs coexist with BK and SK channels in inferior olivary (IO) neurons that send climbing fibers to innervate cerebellar Purkinje cells for the control of motor learning and timing. Ca2+ influx through the dendritic high-threshold voltage-gated Ca2+ channels activates CaCCs, which contribute to membrane repolarization of IO neurons. Loss of TMEM16B expression resulted in the absence of CaCCs in IO neurons, leading to markedly diminished action potential firing of IO neurons in TMEM16B knockout mice. Moreover, these mutant mice exhibited severe cerebellar motor learning deficits. Our findings thus advance the understanding of the neurophysiology of CaCCs and the ionic basis of IO neuron excitability.