Lack of evidence of synaptic contacts by climbing fibre collaterals to basket and stellate cells in developing rat cerebellar cortex

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.

Cocaine- and amphetamine-regulated transcript (CART) peptides have a wide CNS distribution and appear to play an important role in a number of physiological processes including reward and reinforcement, feeding, locomotion, stress responses and perception of pain. We have further investigated the expression of CART peptides in rat cerebellum, using multiple fluorescent antibodies. Dense fibre-like immunofluorescence was observed in the molecular layer of the vestibular cerebellum (paraflocculus and lobes IX and X of the vermis). There was little or no immunofluorescence in any other region of the cerebellum (vermis or hemispheres). The immunofluorescence in lobes IX and X of the vermis showed a parasagittal banding pattern, with one medial and two lateral bands.

We have provided several lines of evidence that CART peptides are expressed by climbing fibres: (1) There is CART peptide immunofluorescence in the inferior olivary complex, the source of climbing fibres. (2) No cerebellar cell bodies were labelled by the CART peptide antibody. (3) The developmental profile of CART peptide expression was consistent with climbing fibre development and finally (4) triple antibody labelling revealed co-expression of CART peptides with VGluT2 (the glutamate transporter at climbing fibre synapses) at boutons innervating calbindin D-28K labelled proximal Purkinje cell dendrites. Analysis of VGluT2 and CART peptide labelling in more detail showed that not all climbing fibres in the vestibular cerebellum expressed CART peptides but those fibres which did express CART peptides were immunofluorescent at all Purkinje cell synapses.

The anatomical, physiological, and behavioral evidence for the involvement of three regions of the cerebellum in oculomotor behavior is reviewed here: (1) the oculomotor vermis and paravermis of lobules V, IV, and VII; (2) the uvula and nodulus; (3) flocculus and ventral parafloccculus. No region of the cerebellum controls eye movements exclusively, but each receives sensory information relevant for the control of multiple systems. An analysis of the microcircuitry suggests how sagittal climbing fiber zones bring visual information to the oculomotor vermis; convey vestibular information to the uvula and nodulus, while optokinetic space is represented in the flocculus. The mossy fiber projections are more heterogeneous. The importance of the inferior olive in modulating Purkinje cell responses is discussed.

The molecular layer of the cerebellar cortex is populated by glial progenitors that express ionotropic glutamate receptors and extend numerous processes among Purkinje cell dendrites. Here, we show that release of glutamate from climbing fiber (CF) axons produces AMPA receptor currents with rapid kinetics in these NG2-immunoreactive glial cells (NG2⁺ cells) in cerebellar slices. NG2⁺ cells may receive up to 70 discrete inputs from one CF and, unlike mature Purkinje cells, are often innervated by multiple CFs. Paired Purkinje cell-NG2⁺ cell recordings show that one CF can innervate both cell types. CF boutons make direct synaptic junctions with NG2⁺ cell processes, indicating that this rapid neuron-glia signaling occurs at discrete sites rather than through ectopic release at CF-Purkinje cell synapses. This robust activation of Ca²⁺-permeable AMPA receptors in NG2⁺ cells expands the influence of the olivocerebellar projection to this abundant class of glial progenitors.

The vestibular nuclei and posterior cerebellum are the destination of vestibular primary afferents and the subject of this review. The vestibular nuclei include four major nuclei (medial, descending, superior and lateral). In addition, smaller vestibular nuclei include: Y-group, parasolitary nucleus, and nucleus intercalatus. Each of the major nuclei can be subdivided further based primarily on cytological and immunohistochemical histological criteria or differences in afferent and/or efferent projections. The primary afferent projections of vestibular end organs are distributed to several ipsilateral vestibular nuclei. Vestibular nuclei communicate bilaterally through a commissural system that is predominantly inhibitory. Secondary vestibular neurons also receive convergent sensory information from optokinetic circuitry, central visual system and neck proprioceptive systems. Secondary vestibular neurons cannot distinguish between sources of afferent activity. However, the discharge of secondary vestibular neurons can distinguish between “active” and “passive” movements.

The posterior cerebellum has extensive afferent and efferent connections with vestibular nuclei. Vestibular primary afferents are distributed to the ipsilateral uvula-nodulus as mossy fibers. Vestibular secondary afferents are distributed bilaterally. Climbing fibers to the cerebellum originate from two subnuclei of the contralateral inferior olive; the dorsomedial cell column and β-nucleus. Vestibular climbing fibers carry information only from the vertical semicircular canals and otoliths. They establish a coordinate map, arrayed in sagittal zones on the surface of the uvula-nodulus. Purkinje cells respond to vestibular stimulation with antiphasic modulation of climbing fiber responses (CFRs) and simple spikes (SSs). The modulation of SSs is out of phase with the modulation of vestibular primary afferents. Modulation of SSs persists, even after vestibular primary afferents are destroyed by a unilateral labyrinthectomy, suggesting that an interneuronal network, triggered by CFRs is responsible for SS modulation. The vestibulo-cerebellum, imposes a vestibular coordinate system on postural responses and permits adaptive guidance of movement.