Vadim Yakhnitsa

Functions of Interneurons in Mouse Cerebellum

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.

Cerebellar Climbing Fibers Modulate Simple Spikes in Purkinje Cells

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 β-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.

Antiphasic Purkinje cell responses in mouse uvula-nodulus are sensitive to static roll–tilt and topographically organized

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.

Microlesions of the Inferior Olive Reduce Vestibular Modulation of Purkinje Cell Complex and Simple Spikes in Mouse Cerebellum

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.

Climbing fibers induce microRNA transcription in cerebellar Purkinje cells.

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.

Topsy Turvy: Functions of Climbing and Mossy Fibers in the Vestibulo-Cerebellum

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.

Activity-Dependent Expression of Acyl-Coenzyme A-Binding Protein in Retinal Muller Glial Cells Evoked by Optokinetic Stimulation

Long-term horizontal optokinetic stimulation (HOKS) decreases the gain of the horizontal optokinetic reflex and evokes the second phase of optokinetic afternystagmus (OKAN-II). We investigated the possible molecular constituents of this adaptation. We used a differential display reverse transcriptase-PCR screen for mRNAs isolated from retinas of rabbits that received HOKS. In each rabbit, we compared mRNAs from the retina stimulated in the posterior→anterior (preferred) direction with mRNAs from the retina stimulated in the anterior→posterior (null) direction. Acyl-CoA-binding protein (ACBP) mRNA was one of four mRNAs selected by this screen, the proteins of which interact with GABA receptors. HOKS in the preferred direction increased ACBP mRNA transcription and ACBP protein expression. ACBP was localized to Muller glial cells by hybridization histochemistry and by immunohistochemistry. ACBP interacts with the α1-subunit of the GABAA receptor, as determined by a yeast two-hybrid technique. This interaction was confirmed by coimmunoprecipitation of ACBP and the α1-subunit of the GABAA receptor using an antibody to GABAAα1. The interaction was also confirmed by a “pull-down” assay in which histidine-tagged ACBP was used to pull down the GABAAα1. ACBP does not cross the blood–brain barrier. However, smaller truncated proteolytic fragments of ACBP do, increasing the excitability of central cortical neurons. Muller cells may secrete ACBP in the inner plexiform layer, thereby decreasing the sensitivity of GABAA receptors expressed on the surface of ganglion cell dendrites. Because retinal directional sensitivity is linked to GABAergic transmission, HOKS-induced expression of ACBP could provide a molecular basis for adaptation to HOKS and for the genesis of OKAN-II.

Vestibularly Evoked Climbing-Fiber Responses Modulate Simple Spikes in Rabbit Cerebellar Purkinje Neurons

Abstract: The nodulus receives a primary vestibular afferent input from the ipsilateral labyrinth and a vestibularly related climbing‐fiber input originating from the contralateral labyrinth. Previously we demonstrated that increased discharge of vestibularly evoked climbing‐fiber responses (CFRs) in nodular Purkinje cells was correlated with decreased discharge of simple spikes (SSs). This left unresolved the question of whether vestibularly evoked antiphasic behavior of CFRs and SSs reflects a common neural mechanism or the activation of two separate parallel pathways. We answered this question using natural vestibular stimulation to modulate the discharge of uvula‐nodular Purkinje cells recorded extracellularly in unilaterally labyrinthectomized, chloralose urethane‐anesthetized rabbits. In such animals, vestibular primary afferents projecting to the uvula‐nodulus as mossy fibers remained intact on the side contralateral to the unilateral labyrinthectomy. The discharge of CFRs recorded in ipsilateral nodular Purkinje cells was increased by ipsilateral roll‐tilt while the discharge of SSs was increased by contralateral roll‐tilt. These polarities were reversed for Purkinje cells recorded in the contralateral uvula‐nodulus. The polarity of SS discharge recorded from Purkinje cells on both sides of the nodulus was opposite to that of the vestibular primary mossy‐fiber afferents. SSs continued to respond to contralateral roll‐tilt even when the primary vestibular afferent mossy‐fiber pathway was destroyed by the unilateral labyrinthectomy. Although the discharge of SSs recorded in the contralateral uvula‐nodulus was increased by contralateral roll‐tilt, this modulation was reduced relative to that observed in Purkinje cells recorded in the ipsilateral uvula‐nodulus. We conclude that vestibularly evoked CFRs caused the modulation of SS discharge.

Climbing fiber activity reduces 14-3-3-θ regulated GABAA receptor phosphorylation in cerebellar Purkinje cells

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.

Distribution of granule cells projecting to focal Purkinje cells in mouse uvula-nodulus.

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.