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Climbing and mossy fibers comprise two distinct afferent paths to the cerebellum. Climbing fibers directly evoke a large multispiked action potential in Purkinje cells termed a "complex spike" (CS). By logical exclusion, the other class of Purkinje cell action potential, termed "simple spike" (SS), has often been attributed to activity conveyed by mossy fibers and relayed to Purkinje cells through granule cells. Here, we investigate the relative importance of climbing and mossy fiber pathways in modulating neuronal activity by recording extracellularly from Purkinje cells, as well as from mossy fiber terminals and interneurons in folia 8-10. Sinusoidal roll-tilt vestibular stimulation vigorously modulates the discharge of climbing and mossy fiber afferents, Purkinje cells, and interneurons in folia 9-10 in anesthetized mice. Roll-tilt onto the side ipsilateral to the recording site increases the discharge of both climbing fibers (CSs) and mossy fibers. However, the discharges of SSs decrease during ipsilateral roll-tilt. Unilateral microlesions of the beta nucleus (β-nucleus) of the inferior olive blocks vestibular modulation of both CSs and SSs in contralateral Purkinje cells. The blockage of SSs occurs even though primary and secondary vestibular mossy fibers remain intact. When mossy fiber afferents are damaged by a unilateral labyrinthectomy (UL), vestibular modulation of SSs in Purkinje cells ipsilateral to the UL remains intact. Two inhibitory interneurons, Golgi and stellate cells, could potentially contribute to climbing fiber-induced modulation of SSs. However, during sinusoidal roll-tilt, only stellate cells discharge appropriately out of phase with the discharge of SSs. Golgi cells discharge in phase with SSs. When the vestibularly modulated discharge is blocked by a microlesion of the inferior olive, the modulated discharge of CSs and SSs is also blocked. When the vestibular mossy fiber pathway is destroyed, vestibular modulation of ipsilateral CSs and SSs persists. We conclude that climbing fibers are primarily responsible for the vestibularly modulated discharge of both CSs and SSs. Modulation of the discharge of SSs is likely caused by climbing fiber-evoked stellate cell inhibition.

The effort to determine morphological and anatomically defined neuronal characteristics from extracellularly recorded physiological signatures has been attempted with varying success in different brain areas. Recent studies have attempted such classification of cerebellar interneurons (CINs) based on statistical measures of spontaneous activity. Previously, such efforts in different brain areas have used supervised clustering methods based on standard parameterizations of spontaneous interspike interval (ISI) histograms. We worried that this might bias researchers toward positive identification results and decided to take a different approach. We recorded CINs from anesthetized cats. We used unsupervised clustering methods applied to a nonparametric representation of the ISI histograms to identify groups of CINs with similar spontaneous activity and then asked how these groups map onto different cell types. Our approach was a fuzzy C-means clustering algorithm applied to the Kullbach-Leibler distances between ISI histograms. We found that there is, in fact, a natural clustering of the spontaneous activity of CINs into six groups but that there was no relationship between this clustering and the standard morphologically defined cell types. These results proved robust when generalization was tested to completely new datasets, including datasets recorded under different anesthesia conditions and in different laboratories and different species (rats). Our results suggest the importance of an unsupervised approach in categorizing neurons according to their extracellular activity. Indeed, a reexamination of such categorization efforts throughout the brain may be necessary. One important open question is that of functional differences of our six spontaneously defined clusters during actual behavior., (Copyright © 2015 the authors 0270-6474/15/351432-11$15.00/0.)

Cerebellar Purkinje cells are excited by two afferent pathways: climbing and mossy fibers. Climbing fibers evoke large "complex spikes" (CSs) that discharge at low frequencies. Mossy fibers synapse on granule cells whose parallel fibers excite Purkinje cells and may contribute to the genesis of "simple spikes" (SSs). Both afferent systems convey vestibular information to folia 9c-10. After making a unilateral labyrinthectomy (UL) in mice, we tested how the discharge of CSs and SSs was changed by the loss of primary vestibular afferent mossy fibers during sinusoidal roll tilt. We recorded from cells identified by juxtacellular neurobiotin labeling. The UL preferentially reduced vestibular modulation of CSs and SSs in folia 8-10 contralateral to the UL. The effects of a UL on Purkinje cell discharge were similar in folia 9c-10, to which vestibular primary afferents project, and in folia 8-9a, to which they do not project, suggesting that vestibular primary afferent mossy fibers were not responsible for the UL-induced alteration of SS discharge. UL also induced reduced vestibular modulation of stellate cell discharge contralateral to the UL. We attribute the decreased modulation to reduced vestibular modulation of climbing fibers. In summary, climbing fibers modulate CSs directly and SSs indirectly through activation of stellate cells. Whereas vestibular primary afferent mossy fibers cannot account for the modulated discharge of SSs or stellate cells, the nonspecific excitation of Purkinje cells by parallel fibers may set an operating point about which the discharges of SSs are sculpted by climbing fibers.

Cerebellar adaptive plasticity regulates posture and movement in response to changing conditions of sensory stimulation. Study of adaptive plasticity of cerebellar circuitry in vitro confines experimental interest to mechanisms with a time scale of minutes. However, cerebellar plasticity, measured behaviorally or electrophysiologically in vivo, occurs over a time scale of tens of minutes and hours. Here we investigate how optokinetically-evoked increases in climbing fiber activity influence expression of key subcellular signaling proteins that regulate the accumulation of GABA(A) receptors (GABA(A)Rs) in the cytoplasm of Purkinje cells and their insertion into the plasma membrane. We used long-term horizontal optokinetic stimulation (HOKS) to activate climbing fibers that project to the flocculus of mice. Although long-term increases in climbing fiber activity in vivo do not alter the expression of any of the subunits of GABA(A)Rs expressed by Purkinje cells, they do influence other subcellular events such as transcription and interaction of signaling proteins. Specifically, increased climbing fiber activity evoked decreased expression of 14-3-3-θ, reduced serine phosphorylation of GABA(A)g(2), and reduced the interaction of 14-3-3-θ with protein kinase C-γ (PKC-γ). Knockdown of 14-3-3-θ in vivo reduced the serine phosphorylation of GABA(A)γ(2). Conversely, treatment of cerebellar lysates with phorbol 12-myristate-13-acetate (PMA), a PKC activator, increased serine phosphorylation of GABA(A)γ(2). Knockdown of 14-3-3-θ or PKC-γ in N2a cells in vitro reduced serine phosphorylation of GABA(A)γ(2) and reduced its cell-surface expression. We interpret these data to mean that a prolonged increase in climbing fiber activity decreases the cell-surface expression of GABA(A)Rs in Purkinje cells and thereby reduces their sensitivity to GABAergic inhibition. This provides a homeostatic mechanism by which Purkinje cells become less sensitive to stellate cell inhibition also evoked by climbing fiber activity., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cerebellar Purkinje cells have two distinct action potentials: complex spikes (CSs) are evoked by single climbing fibers that originate from the contralateral inferior olive. Simple spikes (SSs) are often ascribed to mossy fiber-granule cell-parallel fiber inputs to Purkinje cells. Although generally accepted, this view lacks experimental support. Vestibular stimulation independently activates primary afferent mossy fibers and tertiary afferent climbing fibers that project to the uvula-nodulus (folia 8-10). CSs and SSs normally discharge antiphasically during sinusoidal roll-tilt. When CSs increase, SSs decrease. We tested the relative independence of these pathways in mice by making electrolytic microlesions of the two inferior olivary nuclei from which vestibular climbing fibers originate; the β-nucleus and dorsomedial cell column. This reduced vestibular climbing fiber signaling to the contralateral folia 8-10, while leaving intact vestibular primary and secondary afferent mossy fibers. We recorded from Purkinje cells and interneurons in folia 8-10, identified by juxtacellular labeling with Neurobiotin. Microlesions of the inferior olive increased the spontaneous discharge of SSs in contralateral folia 8-10, but blocked their modulation during vestibular stimulation. The vestibularly evoked discharge of excitatory cerebellar interneurons (granule cells and unipolar brush cells) was not modified by olivary microlesions. The modulated discharge of stellate cells, but not Golgi cells, was reduced by olivary microlesions. We conclude that vestibular modulation of CSs and SSs depends on intact climbing fibers. The absence of vestibularly modulated SSs following olivary microlesions reflects the loss of climbing fiber-evoked stellate cell discharge.

The cerebellum's role in sensory-motor control and adaptation is undisputed. However, a key hypothesis pertaining to the function of cerebellar circuitry lacks experimental support. It is universally assumed that the discharge of mossy fibers accounts for modulation of Purkinje cell "simple spikes" (SSs). This assumption acts as a prism through which all other functions of cerebellar circuitry are viewed. The vestibulo-cerebellum (nodulus and uvula) receives a large, unilateral, vestibular primary afferent mossy fiber projection. We can test its role in modulating Purkinje cell SSs by recording the modulated activity of both mossy fiber terminals and Purkinje cell SSs evoked by identical natural vestibular stimulation. Sinusoidal rotation about the longitudinal axis (roll) modulates the activity of vestibular primary afferent mossy and climbing fibers as well as Purkinje cell SSs and complex spikes (CSs). Remarkably, vestibular primary afferent mossy fibers discharge 180 degrees out of phase with SSs. This indicates that mossy fibers cannot account for SS modulation unless an inhibitory synapse is interposed between mossy fibers or vestibular climbing fibers and Purkinje cells. The authors review several experiments that address the relative contributions of mossy and climbing fiber afferents to the modulation of SSs. They conclude that climbing fibers, not mossy fibers, are primarily responsible for the modulation of SSs as well as CSs and they propose revised functions for these two afferent systems.

Mossy and climbing fibers convey a broad array of signals from vestibular end organs to Purkinje cells in the vestibulo-cerebellum. We have shown previously that Purkinje cell simple spikes (SSs) and climbing fiber-evoked complex spikes (CSs) in the mouse uvula-nodulus are arrayed in 400 microm wide sagittal climbing fiber zones corresponding to the rotational axes of the vertical semicircular canals. It is often assumed that mossy fibers modulate a higher frequency of SSs through the intermediary action of granule cells whose parallel fibers course through the Purkinje cell dendritic tree. This assumption is complicated by the diffuse topography of vestibular primary afferent mossy fiber projections to the uvula-nodulus and the dispersion of mossy fiber signals along folial axes by parallel fibers. Here we measure this parallel fiber dispersion. We made microinjections of neurobiotin into the molecular layers of different folia within the mouse vestibulo-cerebellum and measured the distribution of granule cells retrogradely labeled by the injected neurobiotin. Sixty-two percent of labeled granule cells were located outside a 400 microm sagittal zone flanking the injection site. The dispersion of labeled granule cells was approximately 2.5 mm along folial axes that were 2.7-2.9 mm wide. Our data suggest that topographic specificity of SSs could not be attributed to the topography of vestibular primary afferent mossy fiber-granule cell projections. Rather the response specificity of SSs must be attributed to other mechanisms related to climbing fiber-evoked Purkinje cell and interneuronal activity.

The output signal of Purkinje cells is conveyed by the modulated discharge of simple spikes (SSs) often ascribed to mossy fiber-granule cell-parallel fiber inputs to Purkinje cell dendrites. Although generally accepted, this view lacks experimental support. We can address this view by controlling afferent signals that reach the cerebellum over climbing and mossy fiber pathways. Vestibular primary afferents constitute the largest mossy fiber projection to the uvula-nodulus. The discharge of vestibular primary afferent mossy fibers increases during ipsilateral roll tilt. The discharge of SSs decreases during ipsilateral roll tilt. Climbing fiber discharge [complex spikes (CSs)] increases during ipsilateral roll tilt. These observations suggest that the modulation of SSs during vestibular stimulation cannot be attributed directly to vestibular mossy fiber afferents. Rather we suggest that interneurons driven by vestibular climbing fibers may determine SS modulation. We recorded from cerebellar interneurons (granule, unipolar brush, Golgi, stellate, basket, and Lugaro cells) and Purkinje cells in the uvula-nodulus of anesthetized mice during vestibular stimulation. We identified all neuronal types by juxtacellular labeling with neurobiotin. Granule, unipolar brush, stellate, and basket cells discharge in phase with ipsilateral roll tilt and in phase with CSs. Golgi cells discharge out of phase with ipsilateral roll tilt and out of phase with CSs. The phases of stellate and basket cell discharge suggests that their activity could account for the antiphasic behavior of CSs and SSs. Because Golgi cells discharge in phase with SSs, Golgi cell activity cannot account for SS modulation. The sagittal array of Golgi cell axon terminals suggests that they contribute to the organization of discrete parasagittal vestibular zones.

Two vestibular pathways converge at the uvula-nodulus to modulate the discharge of Purkinje cell complex and simple spikes (CSs and SSs). In the mouse, vestibular primary afferent mossy fibers originate from each of the end organs of the ipsilateral labyrinth and terminate in the granule cell layers of folia 9c-10. Vestibular climbing fiber projections originate from the contralateral beta-nucleus and dorsomedial cell column (dmcc) and terminate directly on Purkinje cells. CSs and SSs could be regulated independently or they could be co-dependent. Here we examine how the discharges of CSs and SSs are modulated by sinusoidal and static roll-tilt in the uvula-nodulus of mice anesthetized with either chloralose-urethane or ketamine-xylazine. All vestibularly-driven CSs and SSs were sensitive to static roll-tilt. None were sensitive to horizontal vestibular stimulation. CSs were modulated in phase with ipsilateral roll-tilt. SSs were modulated out of phase. Spontaneous discharges of CSs were followed by a pause in SSs. Phase leads of CSs and SSs with respect to sinusoidal roll-tilt were advanced by ketamine-xylazine anesthesia relative to chloralose-urethane anesthesia by approximately 45 degrees. The antiphasic modulation of CSs and SSs was independent of anesthetic. Chloralose-urethane, but not ketamine-xylazine, induced spontaneous oscillations of CSs and SSs in 16% of Purkinje cells. Optimal planes of CSs in folia 9c-10 Purkinje cells were organized topographically into sagittal zones whose widths were approximately 400 microm. Purkinje cells with optimal planes in the posterior quadrant of the ipsilateral hemi-field were located in a medial zone. Purkinje cells with optimal planes in the anterior quadrant of the ipsilateral hemi-field were located in a lateral zone. The CS-associated pause in SSs establishes a vector-specific SS output. The amplitude of SS modulation may depend on parallel fiber-mediated signals to Purkinje cells as well as on the state of cerebellar interneurons.

Purkinje cells have two action potentials: Climbing fiber responses (CFRs) and simple spikes (SSs). CFRs reflect the discharge of a single climbing fiber at multiple synaptic sites on the proximal dendrite of the Purkinje cell. SSs reflect the summed action of a subset of parallel fiber synapses on Purkinje cell dendritic spines. Because mossy fiber afferents terminate on granule cells, the ascending axons of which bifurcate, giving rise to parallel fibers, the modulation of SSs has been attributed to mossy fiber afferent signals. This inference has never been tested. Conversely, the low discharge frequency of CFRs has led many to conclude that they have a unique and intermittent role in cerebellar signal processing. We examine the relative potency of vestibularly modulated mossy fiber and climbing fiber signals in evoking CFRs and SSs in Purkinje cells of the uvula-nodulus in chloralose-urethane-anesthetized rabbits. Vestibular primary afferents were blocked by unilateral labyrinthectomy (UL). A UL destroys the vestibular primary afferent signal to the ipsilateral uvula-nodulus, while leaving intact the vestibular climbing fiber signal from the contralateral inferior olive. After UL, vestibular stimulation modulated CFRs and SSs in ipsilateral uvula-nodular Purkinje cells, demonstrating that the primary vestibular afferent mossy fiber input to the ipsilateral uvula-nodulus was not necessary for SS modulation. Unilateral microlesions of the caudal half of the beta-nucleus of the inferior olive reduced a modulated climbing fiber signal to the contralateral uvula-nodulus, causing loss of both vestibularly modulated CFRs and SSs in contralateral Purkinje cells. Vestibular climbing fibers not only evoke low-frequency CFRs, but also indirectly modulate higher-frequency SSs. This modulation must be attributed to cerebellar interneurons. Golgi cell inhibition of granule cells may provide the interneuronal mechanism for CFR-induced SS modulation.

Natural vestibular and optokinetic stimulation were used to investigate the possible role of the cerebellar nodulus in the regulation and modification of reflexive eye movements in rabbits. The nodulus and folium 9d of the uvula were destroyed by surgical aspiration. Before and after nodulectomy the vertical and horizontal vestibuloocular reflexes (VVOR, HVOR) were measured during sinusoidal vestibular stimulation about the longitudinal (roll) and vertical (yaw) axes. Although the gain of the HVOR (G(HVOR) = peak eye movement velocity/peak head velocity) was not affected by the nodulectomy, the gain of the VVOR (G(VVOR)) was reduced. The gains of the vertical and horizontal optokinetic reflexes (G(VOKR), G(HOKR)) were measured during monocular, sinusoidal optokinetic stimulation (OKS) about the longitudinal and vertical axes. Following nodulectomy, there was no reduction in G(VOKR) or G(HOKR). Long-term binocular OKS was used to generate optokinetic afternystagmus, OKAN II, that lasts for hours. After OKAN II was induced, rabbits were subjected to static pitch and roll, to determine how the plane and velocity of OKAN II is influenced by a changing vestibular environment. During static pitch, OKAN II slow phase remained aligned with earth-horizontal. This was true for normal and nodulectomized rabbits. During static roll, OKAN II remained aligned with earth-horizontal in normal rabbits. During static roll in nodulectomized rabbits, OKAN II slow phase developed a centripetal vertical drift. We examined the suppression and recovery of G(VVOR) following exposure to conflicting vertical OKS for 10-30 min. This vestibular-optokinetic conflict reduced G(VVOR) in both normal and nodulectomized rabbits. The time course of recovery of G(VVOR) after conflicting OKS was the same before and after nodulectomy. In normal rabbits, the head pitch angle, at which peak OKAN II velocity occurred, corresponded to the head pitch angle maintained during long-term OKS. If the head was maintained in a "pitched-up" or "pitched-down" orientation during long-term OKS, the subsequently measured OKAN II peak velocity occurred at the same orientation. This was not true for nodulectomized rabbits, who had OKAN II peak velocities at head pitch angles independent of those maintained during long-term OKS. We conclude that the nodulus participates in the regulation of compensatory reflexive movements. The nodulus also influences "remembered" head position in space derived from previous optokinetic and vestibular stimulation.

Abstract: The coordinated expression of as many as 100 proteins may be required to sustain simple changes in synaptic transmission. While each protein may be regulated separately, the translation of multiple proteins could be regulated by microRNAs. MicroRNAs are short non-coding RNAs that translationally repress cognate sequences in targeted mRNAs. If these targeted sequences are shared across several genes, then a single microRNA could, effectively regulate the activity of several genes in parallel. Here we investigate whether microRNA transcription is influenced by naturally evoked synaptic activity at the climbing fiber-Purkinje cell synapse in the mouse cerebellar flocculus. Mice received 24 h of binocular horizontal optokinetic stimulation (HOKS) evoking sustained increases in climbing fiber activity to Purkinje cells in one flocculus and decreases to Purkinje cells in the other. Increased climbing fiber activity increased transcription of 12 microRNAs in the flocculus. The transcription of one of these microRNAs, miR335, was proportional to duration of stimulation, increasing 18-fold after 24 h of HOKS. We localized miR335 transcripts to Purkinje cells using hybridization histochemistry. Transcripts of miR335 decayed to baseline within 3 h after HOKS was stopped. We identified mRNA targets for miR335 using multiple screens: sequence analysis, microinjection of miR335 inhibitors and identification of mRNAs whose transcription decreased during HOKS. Two genes, calbindin and 14–3–3–θ passed these screens. Our data suggest that microRNA transcription could provide an important synaptic or homeostatic mechanism for the regulation of proteins that contribute to Purkinje cell plasticity. [ABSTRACT FROM AUTHOR]