Biomedical subjects

C I De Zeeuw

Publications and source records attributed to C I De Zeeuw.

At least 19 recordsLinked to original sources

PubMed2006

Echinoderm microtubule-associated protein like protein 4, a member of the echinoderm microtubule-associated protein family, stabilizes microtubules.

Echinoderm microtubule-associated protein (EMAP) is the major microtubule binding protein in dividing sea urchin (Strongylocentrotus purpuratus) eggs. Echinoderm microtubule-associated protein like protein 4 (Eml4, restrictedly overexpressed proliferation-associated protein 120 kDa (Ropp120)) is one of the five mammalian EMAP homologues, the cellular function of which remains to be elucidated. In our first set of experiments we determined the spatio-temporal expression pattern of Eml4 in mouse brain. Our results demonstrate that Eml4 is a highly developmentally regulated gene with high expression levels in the developing nervous system of E11 embryos declining to low levels in adult. Spatially, Eml4 expression becomes restricted to the olfactory bulb, hippocampus and cerebellum. Transient transfection of a fusion construct of full-length mouse Eml4 with green fluorescent protein (GFP-Eml4) into Cos7 and HeLa cells resulted in colocalization of GFP-Eml4 with microtubules. This colocalization was observed both with microtubules of non-dividing cells and with the mitotic spindle of dividing cells. In addition, transient overexpression of GFP-Eml4 in Cos7 cells resulted in microtubules that were resistant to nocodazole treatment suggesting that Eml4 stabilizes microtubules. A consequence of microtubule stabilization is a net reduction in the amount of free tubulin. Microtubule stabilizing proteins therefore are expected to indirectly decrease the microtubule growth rate. Indeed, transient transfection of GFP-Eml4 resulted in a marked decrease in the microtubule growth rate, which is in line with our hypothesis that Eml4 functions as a microtubule stabilizing protein. In summary, our results suggest that Eml4 is a developmentally regulated protein that colocalizes with and stabilizes microtubules.

Animals↗

PubMed2006

Oculomotor plasticity during vestibular compensation does not depend on cerebellar LTD.

Vestibular paradigms are widely used for investigating mechanisms underlying cerebellar motor learning. These include adaptation of the vestibuloocular reflex (VOR) after visual-vestibular mismatch training and vestibular compensation after unilateral damage to the vestibular apparatus. To date, various studies have shown that VOR adaptation may be supported by long-term depression (LTD) at the parallel fiber to Purkinje cell synapse. Yet it is unknown to what extent vestibular compensation may depend on this cellular process. Here we investigated adaptive gain changes in the VOR and optokinetic reflex during vestibular compensation in transgenic mice in which LTD is specifically blocked in Purkinje cells via expression of a peptide inhibitor of protein kinase C (L7-PKCi mutants). The results demonstrate that neither the strength nor the time course of vestibular compensation are affected by the absence of LTD. In contrast, analysis of vestibular compensation in spontaneous mutants that lack a functional olivo-cerebellar circuit (lurchers) shows that this form of motor learning is severely impaired. We conclude that oculomotor plasticity during vestibular compensation depends critically on intact cerebellar circuitry but not on the occurrence of cerebellar LTD.

Animals↗

PubMed2005

Deletion of FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, and attenuates cerebellar eyelid conditioning in Fragile X syndrome.

Absence of functional FMRP causes Fragile X syndrome. Abnormalities in synaptic processes in the cerebral cortex and hippocampus contribute to cognitive deficits in Fragile X patients. So far, the potential roles of cerebellar deficits have not been investigated. Here, we demonstrate that both global and Purkinje cell-specific knockouts of Fmr1 show deficits in classical delay eye-blink conditioning in that the percentage of conditioned responses as well as their peak amplitude and peak velocity are reduced. Purkinje cells of these mice show elongated spines and enhanced LTD induction at the parallel fiber synapses that innervate these spines. Moreover, Fragile X patients display the same cerebellar deficits in eye-blink conditioning as the mutant mice. These data indicate that a lack of FMRP leads to cerebellar deficits at both the cellular and behavioral levels and raise the possibility that cerebellar dysfunctions can contribute to motor learning deficits in Fragile X patients.

Animals↗

PubMed2005

GATA3 haploinsufficiency causes a rapid deterioration of distortion product otoacoustic emissions (DPOAEs) in mice.

Human HDR (hypoparathyroidism, deafness and renal dysplasia)-syndrome is caused by haploinsufficiency of zinc-finger transcription factor GATA3. The hearing loss due to GATA3 haploinsufficiency has been shown to be peripheral in origin, but it is unclear to what extent potential aberrations in the outer hair cells (OHCs) contribute to this disorder. To further elucidate the pathophysiological mechanism underlying the hearing defect in HDR-syndrome, we investigated the OHCs in heterozygous Gata3-knockout mice at both the functional and morphological level. While the signal-to-noise ratios of distortion product otoacoustic emissions (DPOAE) in wild type mice did not change significantly during the first half-year of live, those in the heterozygous Gata3 mice decreased dramatically. In addition, both light microscopic and transmission electron microscopic analyses showed that the number of OHCs containing vacuoles was increased in the mutants. Together, these findings indicate that outer hair cell malfunctioning plays a major role in the hearing loss in HDR-syndrome.

Age Factors↗

PubMed2005

Increased noise level of purkinje cell activities minimizes impact of their modulation during sensorimotor control.

While firing rate is well established as a relevant parameter for encoding information exchanged between neurons, the significance of other parameters is more conjectural. Here, we show that regularity of neuronal spike activities affects sensorimotor processing in tottering mutants, which suffer from a mutation in P/Q-type voltage-gated calcium channels. While the modulation amplitude of the simple spike firing rate of their floccular Purkinje cells during optokinetic stimulation is indistinguishable from that of wild-types, the regularity of their firing is markedly disrupted. The gain and phase values of tottering's compensatory eye movements are indistinguishable from those of flocculectomized wild-types or from totterings with the flocculus treated with P/Q-type calcium channel blockers. Moreover, normal eye movements can be evoked in tottering when the flocculus is electrically stimulated with regular spike trains mimicking the firing pattern of normal simple spikes. This study demonstrates the importance of regularity of firing in Purkinje cells for neuronal information processing.

Action Potentials↗

PubMed2005

Gap junctions synchronize synaptic input rather than spike output of olivary neurons.

Electronic coupling in the inferior olive is supposed to underlie the synchrony of complex spike activities of Purkinje cells in the cerebellar cortex. Here we show a computational model which suggests that the olivary gap junctions may synchronize the input rather than the neuronal output. As such, coupling may influence the absolute moment in time of the complex spike activity rather than their synchrony.

Action Potentials↗

PubMed2005

The cervico-ocular reflex is increased in whiplash injury patients.

Whiplash-associated disorders (WAD) are a major problem in the Western world, which put a formidable financial burden on modern society and which evoke an emerging debate on the true nature of their origin. To date there is no generally accepted test that allows us to diagnose WAD objectively. Because whiplash injury causes dysfunction of proprioception in the neck, we investigated the characteristics of the cervico-ocular reflex (COR) of presumptive WAD patients. These patients and age-matched healthy controls were rotated at different stimulus peak velocities in the dark while their head was fixed in space. The gain values of the COR were significantly increased in the patient population at a wide range of stimulus peak velocities with maximum difference at the lower frequencies (p = 0.037, ANOVA). Hence, although larger numbers of patients should be measured, the COR gain appears to be a parameter that may permit an objective diagnosis of WAD.

Adult↗

PubMed2004

Eml5, a novel WD40 domain protein expressed in rat brain.

We have isolated a novel transcript with homology to the major microtubule-associated protein in dividing sea urchin embryos, EMAP. The protein has a predicted MW of approximately 180 kDa and we have named it Eml5 (EMAP-like protein 5). Eml5 contains 11 putative WD40 domains and 3 hydrophobic stretches of 43 aa, HELP domains, which have been suggested to be involved in microtubule binding. Eml5 appears to consist of two tandem repeats of the complete EMAP protein separated by a putative dimerization domain. Eml5 mRNA and protein is expressed at high levels in the hippocampus, cerebellum and olfactory bulb, as determined by in situ hybridization and immunocytochemistry. Eml5 transcripts can be detected in fore- and hindbrain structures from embryonic day 13 onwards. Because other EMAP-like proteins are involved in regulating microtubule dynamics, it is likely that Eml5 plays a role in the regulation of cytoskeletal rearrangements during neuronal development and in adult brain

Alternative Splicing↗

PubMed2004

Simple spike and complex spike activity of floccular Purkinje cells during the optokinetic reflex in mice lacking cerebellar long-term depression.

Cerebellar long-term depression (LTD) at parallel fibre-Purkinje cell (P-cell) synapses is thought to embody neuronal information storage for motor learning. Transgenic L7-protein kinase C inhibitor (PKCI) mice in which cerebellar LTD is selectively blocked do indeed exhibit impaired adaptation in the vestibulo-ocular reflex (VOR) while their default oculomotor performance is unaffected. Although supportive, these data do not definitively establish a causal link between memory storage required for motor learning and cerebellar LTD. As the L7-PKCI transgene is probably activated from the early stages of P-cell development, an alternative could be that P-cells develop abnormal signals in L7-PKCI mutants, disturbing mechanisms of motor learning that rely on proper P-cell outputs. To test this alternative hypothesis, we studied simple spike (SS) and complex spike (CS) activity of vertical axis P-cells in the flocculus of L7-PKCI mice and their wild-type littermates during sinusoidal optokinetic stimulation. Both SS and CS discharge dynamics appeared to be very similar in wild-type and transgenic P-cells at all stimulus frequencies (0.05-0.8 Hz). The CS activity of all vertical axis cells increased with contralateral stimulus rotation and lagged ipsiversive eye velocity by 165-180 degrees. The SS modulation was roughly reciprocal to the CS modulation and lagged ipsiversive eye velocity by approximately 15 degrees. The baseline SS and CS discharge characteristics were indistinguishable between the two genotypes. We conclude that the impaired VOR learning in L7-PKCI mutants does not reflect fundamental aberrations of the cerebellar circuitry. The data thus strengthen the evidence that cerebellar LTD is implicated in rapid VOR learning but not in the development of normal default response patterns.

Action Potentials↗

PubMed2003

Cerebellar LTD and learning-dependent timing of conditioned eyelid responses.

Mammals can be trained to make a conditioned movement at a precise time, which is correlated to the interval between the conditioned stimulus and unconditioned stimulus during the learning. This learning-dependent timing has been shown to depend on an intact cerebellar cortex, but which cellular process is responsible for this form of learning remains to be demonstrated. Here, we show that protein kinase C-dependent long-term depression in Purkinje cells is necessary for learning-dependent timing of Pavlovian-conditioned eyeblink responses.

Animals↗

PubMed2002

Cerebellar LTD facilitates but is not essential for long-term adaptation of the vestibulo-ocular reflex.

The induction of cerebellar long-term depression (LTD) at the parallel fibre-Purkinje cell synapse is selectively blocked in L7-PKCi transgenic mice, rendering these mice unable to adaptively modify their vestibulo-ocular reflex (VOR) during visuo-vestibular training for a few hours. Despite this deficit, their eye movement performance as well as their general motor behaviour appears unaffected. This combination suggests that, in the long term, residual forms of plasticity in the vestibulo-cerebellar circuitry can compensate for the absence of cerebellar LTD. To investigate whether LTD-deficient mice exhibit motor learning in the long run, we subjected L7-PKCi transgenic mice to visuo-vestibular training paradigms that were aimed at either increasing or decreasing the VOR response in the course of eight consecutive days. During the increasing paradigm, the VOR gain of transgenic mice increased significantly, while VOR gain decreased and VOR phase-lead increased during the decreasing paradigm. The impact of these long training periods on the VOR was significantly smaller in LTD-deficient mice than in wild type littermates. Thus, while LTD may be necessary for short-term VOR adaptation, it facilitates but is not required for long-term adaptation of the VOR.

Adaptation, Physiological↗

PubMed2002

Analysis of Cx36 knockout does not support tenet that olivary gap junctions are required for complex spike synchronization and normal motor performance.

Electrotonic coupling by gap junctions between neurons in the inferior olive has been claimed to underly complex spike (CS) synchrony of Purkinje cells in the cerebellar cortex and thereby to play a role in the coordination of movements. Here, we investigated the motor performance of mice that lack connexin36 (Cx36), which appears necessary for functional olivary gap junctions. Cx36 null-mutants are not ataxic, they show a normal performance on the accelerating rotorod, and they have a regular walking pattern. In addition, they show normal compensatory eye movements during sinusoidal visual and/or vestibular stimulation. To find out whether the normal motor performance in mutants reflects normal CS activity or some compensatory mechanism downstream of the cerebellar cortex, we determined the CS firing rate, climbing-fiber pause, and degree of CS synchrony. None of these parameters in the mutants differed from those in wildtype littermates. Finally, we investigated whether the role of coupling becomes apparent under challenging conditions, such as during application of the tremorgenic drug harmaline, which specifically turns olivary neurons into an oscillatory state at a high frequency. In both the mutants and wildtypes this application induced tremors of a similar duration with similar peak frequencies and amplitudes. Thus surprisingly, the present data does not support the notion that electrotonic coupling by gap junctions underlies synchronization of olivary spike activity and that these gap junctions are essential for normal motor performance.

Action Potentials↗

PubMed2002

Motor performance and motor learning in Lurcher mice.

In adult Lurcher mice virtually all cerebellar Purkinje cells have degenerated as a direct consequence of mutant gene action, providing a natural model for studying the effect of cerebellar cortical lesions on the generation of compensatory eye movements. Lurcher mice possess both optokinetic (OKR) and vestibular (VOR) compensatory reflexes. However, clear differences were observed in control of the OKR consisting of a large reduction in gain and a moderate increase in phase lag. Minor differences were also observed in the VOR in that gain and phase lead of the reflex were both increased in Lurcher animals. Subjecting Lurcher animals to eight days of visuovestibular training tested the assumption that increased VOR gain reflected an adaptive mechanism within remaining brainstem oculomotor pathways to compensate for the reduced OKR. Contrary to control animals, Lurcher animals were unable to modify either VOR or OKR in the course of training and therefore confirmed that an intact cerebellum is indispensable for the implementation of adaptive modifications to the oculomotor system.

Animals↗

PubMed2002

Monitoring kinetic and frequency-domain properties of eyelid responses in mice with magnetic distance measurement technique.

Classical eye-blink conditioning in mutant mice can be used to study the molecular mechanisms underlying associative learning. To measure the kinetic and frequency domain properties of conditioned (tone - periorbital shock procedure) and unconditioned eyelid responses in freely moving mice, we developed a method that allows adequate, absolute, and continuous determination of their eyelid movements in time and space while using an electrical shock as the unconditioned stimulus. The basic principle is to generate a local magnetic field that moves with the animal and that is picked up by either a field-sensitive chip or coil. With the use of this magnetic distance measurement technique (MDMT), but not with the use of electromyographic recordings, we were able to measure mean latency, peak amplitude, velocity, and acceleration of unconditioned eyelid responses, which equaled 7.9 +/- 0.2 ms, 1.2 +/- 0.02 mm, 28.5 +/- 1 mm/s, and 637 +/- 22 mm/s(2), respectively (means +/- SD). During conditioning, the mice reached an average of 78% of conditioned responses over four training sessions, while animals that were subjected to randomly paired conditioned and unconditioned stimuli showed no significant increases. The mean latency of the conditioned responses decreased from 222 +/- 40 ms in session 2 to 127 +/- 6 ms in session 4, while their mean peak latency increased from 321 +/- 45 to 416 +/- 67 ms. The mean peak amplitudes, peak velocities, and peak acceleration of these responses increased from 0.62 +/- 0.02 to 0.77 +/- 0.02 mm, from 3.9 +/- 0.3 to 7.7 +/- 0.5 mm/s, and from 81 +/- 7 to 139 +/- 10 mm/s(2), respectively. Power spectra of acceleration records illustrated that both the unconditioned and conditioned responses of mice had oscillatory properties with a dominant peak frequency close to 25 Hz that was not dependent on training session, interstimulus interval, or response size. These data show that MDMT can be used to measure the kinetics and frequency domain properties of conditioned eyelid responses in mice and that these properties follow the dynamic characteristics of other mammals.

Animals↗

PubMed2001

Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes.

Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes.

Animals↗

PubMed2001

Expression of protein kinase C inhibitor blocks cerebellar long-term depression without affecting Purkinje cell excitability in alert mice.

A longstanding but still controversial hypothesis is that long-term depression (LTD) of parallel fiber-Purkinje cell synapses in the cerebellum embodies part of the neuronal information storage required for associative motor learning. Transgenic mice in which LTD is blocked by Purkinje cell-specific inhibition of protein kinase C (PKC) (L7-PKCI mutants) do indeed show impaired adaptation of their vestibulo-ocular reflex, whereas the dynamics of their eye movement performance are unaffected. However, because L7-PKCI mutants have a persistent multiple climbing fiber innervation at least until 35 d of age and because the baseline discharge of the Purkinje cells in the L7-PKCI mutants is unknown, factors other than a blockage of LTD induction itself may underlie their impaired motor learning. We therefore investigated the spontaneous discharge of Purkinje cells in alert adult L7-PKCI mice as well as their multiple climbing fiber innervation beyond the age of 3 months. We found that the simple spike and complex spike-firing properties (such as mean firing rate, interspike interval, and spike count variability), oscillations, and climbing fiber pause in the L7-PKCI mutants were indistinguishable from those in their wild-type littermates. In addition, we found that multiple climbing fiber innervation does not occur in cerebellar slices obtained from 3- to 6-month-old mutants. These data indicate (1) that neither PKC inhibition nor the subsequent blockage of LTD induction disturbs the spontaneous discharge of Purkinje cells in alert mice, (2) that Purkinje cell-specific inhibition of PKC detains rather than prevents the developmental conversion from multiple to mono-innervation of Purkinje cells by climbing fibers, and (3) that as a consequence the impaired motor learning as observed in older adult L7-PKCI mutants cannot be attributable either to a disturbance in the baseline simple spike and complex spike activities of their Purkinje cells or to a persistent multiple climbing fiber innervation. We conclude that cerebellar LTD is probably one of the major mechanisms underlying motor learning, but that deficits in LTD induction and motor learning as observed in the L7-PKCI mutants may only be reflected in differences of the Purkinje cell signals during and/or directly after training.

Action Potentials↗

PubMed2001

Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts.

CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.

3T3 Cells↗

PubMed2001

The dynamic characteristics of the mouse horizontal vestibulo-ocular and optokinetic response.

In the present study the optokinetic reflex, vestibulo-ocular reflex and their interaction were investigated in the mouse, using a modified subconjunctival search coil technique. Gain of the ocular response to sinusoidal optokinetic stimulation was relatively constant for peak velocities lower than 8 degrees /s, ranging from 0.7 to 0.8. Gain decreased proportionally to velocity for faster stimuli. The vestibulo-ocular reflex acted to produce a sinusoidal compensatory eye movement in response to sinusoidal stimuli. The phase of the eye movement with respect to head movement advanced as stimulus frequency decreased, the familiar signature of the torsion pendulum behavior of the semicircular canals. The first-order time constant of the vestibulo-ocular reflex, as measured from the eye velocity decay after a vestibular velocity step, was 660 ms. The response of the vestibulo-ocular reflex changed with stimulus amplitude, having a higher gain and smaller phase lead when stimulus amplitude was increased. As a result of this nonlinear behavior, reflex gain correlated strongly with stimulus acceleration over the 0.1-1.6 Hz frequency range. When whole body rotation was performed in the light the optokinetic and vestibular system combined to generate nearly constant response gain (approximately 0.8) and phase (approximately 0 degrees ) over the tested frequency range of 0.1-1.6 Hz. We conclude that the compensatory eye movements of the mouse are similar to those found in other afoveate mammals, but there are also significant differences, namely shorter apparent time constants of the angular VOR and stronger nonlinearities.

Animals↗