Takehito Okamoto

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.

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.

Lipid signaling in cytosolic phospholipase A2α–cyclooxygenase-2 cascade mediates cerebellar long-term depression and motor learning

In this study, we show the crucial roles of lipid signaling in long-term depression (LTD), that is, synaptic plasticity prevailing in cerebellar Purkinje cells. In mouse brain slices, we found that cPLA2α knockout blocked LTD induction, which was rescued by replenishing arachidonic acid (AA) or prostaglandin (PG) D2 or E2. Moreover, cyclooxygenase (COX)–2 inhibitors block LTD, which is rescued by supplementing PGD2/E2. The blockade or rescue occurs when these reagents are applied within a time window of 5–15 min following the onset of LTD-inducing stimulation. Furthermore, PGD2/E2 facilitates the chemical induction of LTD by a PKC activator but is unable to rescue the LTD blocked by a PKC inhibitor. We conclude that PGD2/E2 mediates LTD jointly with PKC, and suggest possible pathways for their interaction. Finally, we demonstrate in awake mice that cPLA2α deficiency or COX-2 inhibition attenuates short-term adaptation of optokinetic eye movements, supporting the view that LTD underlies motor learning.

Dual involvement of G-substrate in motor learning revealed by gene deletion.

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate–deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10–15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.

Post-training cerebellar cortical activity plays an important role for consolidation of memory of cerebellum-dependent motor learning

Adaptation of mouse horizontal optokinetic response (HOKR) eye movement provides an experimental model for cerebellum-dependent motor learning. Our previous study revealed that the memory trace of HOKR adaptation is initially encoded in the cerebellar flocculus after hours of optokinetic training, and transferred to the vestibular nuclei to be consolidated to long-term motor memory after days of training [28]. To reveal how the cerebellar cortex operates in the transfer of the memory trace of adaptation, we examined the effects of shutdown of the cerebellar cortex after daily training. Three groups of mice received 1h of optokinetic training daily for 4 days, and showed similar amounts of adaptation after the end of 1h of training throughout 4 days. However, in the mice which daily received bilateral floccular muscimol infusion under gas anesthesia in the post-training period, consolidation of memory of the adaptation was markedly impaired, compared with the control mice which daily received bilateral floccular Ringers solution infusions under gas anesthesia or those which daily received only gas anesthesia. These results are consistent with the studies of the effects of inactivation of cerebellar cortex on the consolidation of motor memory of rabbit eyeblink conditioning [2,4,18], and suggest that the post-training cerebellar cortex activity play an important for the consolidation of motor memory of HOKR adaptation.

Impaired Auditory-Vestibular Functions and Behavioral Abnormalities of Slitrk6-Deficient Mice

A recent study revealed that Slitrk6, a transmembrane protein containing a leucine-rich repeat domain, has a critical role in the development of the inner ear neural circuit. However, it is still unknown how the absence of Slitrk6 affects auditory and vestibular functions. In addition, the role of Slitrk6 in regions of the central nervous system, including the dorsal thalamus, has not been addressed. To understand the physiological role of Slitrk6, Slitrk6-knockout (KO) mice were subjected to systematic behavioral analyses including auditory and vestibular function tests. Compared to wild-type mice, the auditory brainstem response (ABR) of Slitrk6-KO mice indicated a mid-frequency range (8–16 kHz) hearing loss and reduction of the first ABR wave. The auditory startle response was also reduced. A vestibulo-ocular reflex (VOR) test showed decreased vertical (head movement–induced) VOR gains and normal horizontal VOR. In an open field test, locomotor activity was reduced; the tendency to be in the center region was increased, but only in the first 5 min of the test, indicating altered adaptive responses to a novel environment. Altered adaptive responses were also found in a hole-board test in which head-dip behavior was increased and advanced. Aside from these abnormalities, no clear abnormalities were noted in the mood, anxiety, learning, spatial memory, or fear memory–related behavioral tests. These results indicate that the Slitrk6-KO mouse can serve as a model of hereditary sensorineural deafness. Furthermore, the altered responses of Slitrk6-KO mice to the novel environment suggest a role of Slitrk6 in some cognitive functions.

Different effects of transcription and translation inhibition on transfer of memory trace of cerebellum-dependent motor learning

When humans perform rhythmic movement of two limbs in the sagittal plane, the “directional principle” appears: that is, the movement in the same direction is easier than the opposite direction. The purpose of this study is to examine the basis of this principle. In the first experiment, we tested the hypothesis that sending separate motor commands to two limbs is important. We compared the difficulty of movement in two conditions: (1) coordination of voluntary movements of ipsilateral hand and foot, and (2) coordination of voluntary movement of the hand and passive movement of the foot. Each condition composed of the “same direction” task and “opposite direction” task. In both conditions the difficulty of movement obeyed the directional principle. In the second experiment, we investigated the influence of kinesthetic afferent from the foot on the stability of the relationships between the positions of hand and foot. Subjects performed voluntary movement of hand with guidance of auditory pace signal, while ignoring the passive movement of ipsilateral foot that was moved by the experimenter in the same or opposite direction to the hand. No difference of the stability appeared depending on the direction of the foot movement. The results of the first and second experiments suggest that the directional principle is not based on sending separate motor commands to two limbs, nor interference of afferent signals from two limbs. Instead, comparing kinesthetic afferent from two limbs seems to be crucial for the stability of movements of two limbs.