Soichi Nagao

Memory trace of motor learning shifts transsynaptically from cerebellar cortex to nuclei for consolidation

Adaptation of ocular reflexes is a prototype of motor learning. While the cerebellum is acknowledged as the critical site for motor learning, the functional differences between the cerebellar cortex and nuclei in motor memory formation are not precisely known. Two different views are proposed: one that the memory is formed within the cerebellar flocculus, and the other that the memory is formed within vestibular nuclei. Here we developed a new paradigm of long-term adaptation of mouse horizontal optokinetic response eye movements and examined the location of its memory trace. We also tested the role of flocculus and inferior olive in long-term adaptation by chronic lesion experiments. Reversible bilateral flocculus shutdown with local application of 0.5 microl-5% lidocaine extinguished the memory trace of day-long adaptation, while it very little affected the memory trace of week-long adaptation. The responsiveness of vestibular nuclei after week-long adaptation was examined by measuring the extracellular field responses to the electrical stimulation of vestibular nerve under trichloroacetaldehyde anesthesia. The amplitudes and slopes of evoked monosynaptic field response (N1) of week-long adapted mice were enhanced around the medial vestibular nucleus compared with those of control mice. Chronic flocculus or inferior olive lesions abolished both day and week-long adaptations. These results suggest that the functional memory trace of short-term adaptation is formed initially within the cerebellar cortex, and later transferred to vestibular nuclei to be consolidated to a long-term memory. Both day and week-long adaptations were markedly depressed when neural nitric oxide was pharmacologically blocked locally and when neuronal nitric oxide synthase was ablated by gene knockout, suggesting that cerebellar long-term depression underlies both acquisition and consolidation of motor memory.

Subdural application of hemoglobin to the cerebellum blocks vestibuloocular reflex adaptation

Vestibuloocular reflex (VOR) was induced by horizontal sinusoidal whole-body rotation in darkness in rabbits and a monkey. One eye was observed through an infrared TV camera. The gain of VOR was adaptively changed when the animal was continuously rotated for 3 h with the observed eye exposed to the screen moving in phase or out of phase with the head. Injection of 0.1 ml saline solution containing 10 microM hemoglobin into the subdural space over the cerebellar flocculus ipsilateral to the observed eye abolished the VOR adaptation. Since hemoglobin absorbs nitric oxide, which mediates synaptic plasticity in the cerebellar cortex, these results support the view that synaptic plasticity of the flocculus plays a key role in the VOR adaptation.

Impaired cerebellar development and function in mice lacking CAPS2, a protein involved in neurotrophin release

Ca2+-dependent activator protein for secretion 2 (CAPS2/CADPS2) is a secretory granule-associated protein that is abundant at the parallel fiber terminals of granule cells in the mouse cerebellum and is involved in the release of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), both of which are required for cerebellar development. The human homolog gene on chromosome 7 is located within susceptibility locus 1 of autism, a disease characterized by several cerebellar morphological abnormalities. Here we report that CAPS2 knock-out mice are deficient in the release of NT-3 and BDNF, and they consequently exhibit suppressed phosphorylation of Trk receptors in the cerebellum; these mice exhibit pronounced impairments in cerebellar development and functions, including neuronal survival, differentiation and migration of postmitotic granule cells, dendritogenesis of Purkinje cells, lobulation between lobules VI and VII, structure and vesicular distribution of parallel fiber–Purkinje cell synapses, paired-pulse facilitation at parallel fiber–Purkinje cell synapses, rotarod motor coordination, and eye movement plasticity in optokinetic training. Increased granule cell death of the external granular layer was noted in lobules VI–VII and IX, in which high BDNF and NT-3 levels are specifically localized during cerebellar development. Therefore, the deficiency of CAPS2 indicates that CAPS2-mediated neurotrophin release is indispensable for normal cerebellar development and functions, including neuronal differentiation and survival, morphogenesis, synaptic function, and motor leaning/control. The possible involvement of the CAPS2 gene in the cerebellar deficits of autistic patients is discussed.

Differences of the primate flocculus and ventral paraflocculus in the mossy and climbing fiber input organization

Potential sources of cerebellar cortical afferent fibers were identified in the vestibular ganglion, medulla oblongata, pons, and cerebellar nucleus of seven anesthetized Macaca fuscata after local injections of wheat germ agglutinin‐conjugated horseradish peroxidase or Fast Blue into the flocculus (FL) or ventral paraflocculus (VP). There were differences in the sources of mossy fibers to the FL and VP. Labeled neurons, after injections into the FL, were located mainly in the ipsilateral vestibular ganglion, bilaterally in the vestibular and prepositus hypoglossal nuclei, nucleus reticularis tegmenti pontis, and the central part of the mesencephalic reticular formation including the raphe nuclei. Labeled neurons were rarely seen in the pontine nuclei after injections into the FL. By contrast, after injections into the VP, numerous labeled neurons were located in the contralateral pontine nuclei, but relatively few in the vestibular nuclei bilaterally. Sources of climbing fibers to the FL and VP were completely contralateral to the injection side. After the injection into the FL and VP, labeled neurons were located in the dorsal cap, ventrolateral outgrowth, and ventral part of the medial accessory olivary nucleus. The projections from these three olivary areas were generally consistent with a zonal pattern of terminations in the FL and VP. The present results are consistent with a hypothesis that the FL is mainly involved in the control of vestibulo‐ocular reflex and that the VP is mainly involved in the control of smooth pursuit eye movements. J. Comp. Neurol. 382:480‐498, 1997.

Anterograde C1ql1 Signaling Is Required in Order to Determine and Maintain a Single-Winner Climbing Fiber in the Mouse Cerebellum

Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.

Different roles of flocculus and ventral paraflocculus for oculomotor control in the primate.

Responses of Purkinje cells were compared in the monkey flocculus and ventral paraflocculus. During vestibuloocular reflex (VOR) eye movements, flocculus Purkinje cells exhibited simple spike modulation related to head velocity, while ventral paraflocculus Purkinje cells exhibited modulation with no such head velocity preference. During smooth tracking of a sinusoidally moving small target, ventral paraflocculus Purkinje cells exhibited simple spike modulation related to target velocity or position, while the flocculus Purkinje cells exhibited a smaller modulation with no such target preference. Visual suppression of the VOR enhanced the simple spike responses in the ventral paraflocculus, but not in the flocculus. These results suggest that the primate flocculus and the ventral paraflocculus control differentially VOR and smooth pursuit eye movements, respectively.

Role of Primate Cerebellar Hemisphere in Voluntary Eye Movement Control Revealed by Lesion Effects

The anatomical connection between the frontal eye field and the cerebellar hemispheric lobule VII (H-VII) suggests a potential role of the hemisphere in voluntary eye movement control. To reveal the involvement of the hemisphere in smooth pursuit and saccade control, we made a unilateral lesion around H-VII and examined its effects in three Macaca fuscata that were trained to pursue visually a small target. To the step (3 degrees)-ramp (5-20 degrees/s) target motion, the monkeys usually showed an initial pursuit eye movement at a latency of 80-140 ms and a small catch-up saccade at 140-220 ms that was followed by a postsaccadic pursuit eye movement that roughly matched the ramp target velocity. After unilateral cerebellar hemispheric lesioning, the initial pursuit eye movements were impaired, and the velocities of the postsaccadic pursuit eye movements decreased. The onsets of 5 degrees visually guided saccades to the stationary target were delayed, and their amplitudes showed a tendency of increased trial-to-trial variability but never became hypo- or hypermetric. Similar tendencies were observed in the onsets and amplitudes of catch-up saccades. The adaptation of open-loop smooth pursuit velocity, tested by a step increase in target velocity for a brief period, was impaired. These lesion effects were recognized in all directions, particularly in the ipsiversive direction. A recovery was observed at 4 wk postlesion for some of these lesion effects. These results suggest that the cerebellar hemispheric region around lobule VII is involved in the control of smooth pursuit and saccadic eye movements.

Location of efferent terminals of the primate flocculus and ventral paraflocculus revealed by anterograde axonal transport methods.

Efferents of the flocculus (FL) and ventral paraflocculus (VP) were examined in seven anesthetized Macaca fuscata by anterograde axonal transport method using wheat germ agglutinin-conjugated horseradish peroxidase or phaseolus vulgaris leucoagglutinin. Several major foci of axon terminals were found in the vestibular nuclear complex and cerebellar nuclei. A difference was seen in the location of efferent terminals between the FL and VP. When the tracer covered the FL, labeled axon terminals were located within the medial and ventrolateral parts of the medial vestibular nucleus, superior vestibular nucleus and y-group. When the tracer covered the VP, labeled axon terminals were located within the caudo-ventral part of posterior interpositus and dentate nuclei, in addition to the medial and ventrolateral parts of the medial vestibular nucleus, superior vestibular nucleus and y-group. Labeled terminals were virtually absent in the basal interstitial nucleus of the cerebellum. On the points of neo- or paleo-cerebellar cortex fiber connections, these results correspond to our previous anatomical observations that the FL received mossy fiber afferents mainly from the vestibular system and nucleus reticularis tegmenti pontis and very little from the pontine nuclei, whereas the VP received mossy afferents mainly from the nucleus reticularis tegmenti pontis and pontine nuclei and very little from the vestibular system. These anatomical observations are consistent with a hypothesis in our previous anatomical and physiological study that the primate FL and VP mediate rather different functional roles in the oculomotor control.

Type 1 Inositol Trisphosphate Receptor Regulates Cerebellar Circuits by Maintaining the Spine Morphology of Purkinje Cells in Adult Mice

The structural maintenance of neural circuits is critical for higher brain functions in adulthood. Although several molecules have been identified as regulators for spine maintenance in hippocampal and cortical neurons, it is poorly understood how Purkinje cell (PC) spines are maintained in the mature cerebellum. Here we show that the calcium channel type 1 inositol trisphosphate receptor (IP3R1) in PCs plays a crucial role in controlling the maintenance of parallel fiber (PF)–PC synaptic circuits in the mature cerebellum in vivo. Significantly, adult mice lacking IP3R1 specifically in PCs (L7-Cre;Itpr1flox/flox) showed dramatic increase in spine density and spine length of PCs, despite having normal spines during development. In addition, the abnormally rearranged PF–PC synaptic circuits in mature cerebellum caused unexpectedly severe ataxia in adult L7-Cre;Itpr1flox/flox mice. Our findings reveal a specific role for IP3R1 in PCs not only as an intracellular mediator of cerebellar synaptic plasticity induction, but also as a critical regulator of PF–PC synaptic circuit maintenance in the mature cerebellum in vivo; this mechanism may underlie motor coordination and learning in adults.

Role of Cerebellar Cortical Protein Synthesis in Transfer of Memory Trace of Cerebellum-Dependent Motor Learning

We developed a new protocol that induces long-term adaptation of horizontal optokinetic response (HOKR) eye movement by hours of spaced training and examined the role of protein synthesis in the cerebellar cortex in the formation of memory of adaptation. Mice were trained to view 800 cycles of screen oscillation either by 1 h of massed training or by 2.5 h to 8 d of training with 0.5 h to 1 d space intervals. The HOKR gains increased similarly by 20–30% at the end of training; however, the gains increased by 1 h of massed training recovered within 24 h, whereas the gains increased by spaced training were sustained over 24 h. Bilateral floccular lidocaine microinfusions immediately after the end of training recovered the gains increased by 1 h of massed training but did not affect the gains increased by 4 h of spaced training, suggesting that the memory trace of adaptation was transferred from the flocculus to the vestibular nuclei within 4 h of spaced training. Blockade of floccular protein synthesis, examined by bilateral floccular microinfusions of anisomycin or actinomycin D 1–4 h before the training, impaired the gains increased by 4 h of spaced training but did not affect the gains increased by 1 h of massed training. These findings suggest that the transfer of the memory trace of adaptation occurs within 4 h of spaced training, and proteins synthesized in the flocculus during training period may play an important role in memory transfer.