Number and Density of AMPA Receptors in Individual Synapses in the Rat Cerebellum as Revealed by SDS-Digested Freeze-Fracture Replica Labeling

Masugi-Tokita, Miwako; Tarusawa, Etsuko; Watanabe, Masahiko; Molnár, Elek; Fujimoto, Kazushi; Shigemoto, Ryuichi

doi:10.1523/jneurosci.2861-06.2007

Cited by 160 publications

(169 citation statements)

References 55 publications

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“…We questioned whether the reciprocal expression pattern of GluR1 and NR2B was reflected at the level of individual synapses. To label synaptic surface receptors, we used the SDS freeze-fracture replica labeling (SDS-FRL) method (14) with double replica [supporting information (SI) Fig. S1].…”

Section: Resultsmentioning

confidence: 99%

“…S1]. This method allows us to quantitatively detect two different molecules without interference from steric hindrance (14). An excitatory synaptic area is distinguished by the clustering of intramembrane particles (IMPs) on the E-face (14) and positive labeling for NR1 NMDA receptor subunit (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…SR of the left and right CA1 area was trimmed and used for biochemical purification of the PSD fraction. For SDS-FRL, fixed tissues were first frozen by a high-pressure freezing machine and replicated with carbon and platinum (14). After SDS treatment, the replicas were immuno-labeled with antibodies for GluR1 (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors

Shinohara

Hirase

Watanabe

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

181

193

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Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cells are larger and have more perforated PSD and a GluR1 expression level twice as high as those receiving input from the left CA3. The synaptic density of GluR1 increases as the size of a synapse increases, whereas that of NR2B decreases because of the relatively constant NR2B expression in CA1 regardless of synapse size. Densities of other major glutamate receptor subunits show no correlation with synapse size, thus resulting in higher net expression in synapses having right input. Our study demonstrates universal left-right asymmetry of hippocampal synapses with a fundamental relationship between synaptic area and the expression of glutamate receptor subunits.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors

Shinohara

Hirase

Watanabe

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

181

193

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“…1 A and C). First, we measured the density of synaptic AMPARs using SDS-FRL in PF-PC synapses (19) identified with labeling for a mature PF-PC synapse marker, the GluD2 receptor subunit (Fig. 2 A and B) (20).…”

Section: Resultsmentioning

confidence: 99%

“…SDS-FRL was performed with some modifications to the original method described by Fujimoto (29) and was used in our previous study (19). Briefly, under sodium pentobarbital (60 mg/kg) anesthesia, mice were perfused through the ascending aorta with 0.5% paraformaldehyde in 0.1 M sodium phosphate buffer (PB).…”

Section: Methodsmentioning

confidence: 99%

Distinct cerebellar engrams in short-term and long-term motor learning

Wang

Nakadate

Masugi-Tokita

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Cerebellar motor learning is suggested to be caused by long-term plasticity of excitatory parallel fiber-Purkinje cell (PF-PC) synapses associated with changes in the number of synaptic AMPA-type glutamate receptors (AMPARs). However, whether the AMPARs decrease or increase in individual PF-PC synapses occurs in physiological motor learning and accounts for memory that lasts over days remains elusive. We combined quantitative SDS-digested freeze-fracture replica labeling for AMPAR and physical dissector electron microscopy with a simple model of cerebellar motor learning, adaptation of horizontal optokinetic response (HOKR) in mouse. After 1-h training of HOKR, short-term adaptation (STA) was accompanied with transient decrease in AMPARs by 28% in target PF-PC synapses. STA was well correlated with AMPAR decrease in individual animals and both STA and AMPAR decrease recovered to basal levels within 24 h. Surprisingly, long-term adaptation (LTA) after five consecutive daily trainings of 1-h HOKR did not alter the number of AMPARs in PF-PC synapses but caused gradual and persistent synapse elimination by 45%, with corresponding PC spine loss by the fifth training day. Furthermore, recovery of LTA after 2 wk was well correlated with increase of PF-PC synapses to the control level. Our findings indicate that the AMPARs decrease in PF-PC synapses and the elimination of these synapses are in vivo engrams in short-and long-term motor learning, respectively, showing a unique type of synaptic plasticity that may contribute to memory consolidation.long-term depression | high-voltage electron microscope | Golgi staining I mage stabilization in the visual field via the vestibulo-ocular reflex and optokinetic response requires accurate extraocular muscle synergies that rely on long-term plastic calibrations in the cerebellar flocculus (FL) and its downstream target vestibular nuclei (VN) (1-8). Long-term depression (LTD) in parallel fiber-Purkinje cell (PF-PC) synapses has been postulated as a possible mechanism for this plastic calibration based on many lines of mutant mice that lack both LTD and learning (9-12). However, LTD's role in motor learning has been recently questioned by a few mutant mice lines (13) and mice with pharmacological treatments (14) that showed lack of LTD but no impairment of learning. Furthermore, long-term potentiation in PF-PC synapses has been also shown to be involved in the motor learning (15). Recent evidence indicates that various forms of synaptic plasticity works synergistically and can compensate each other when one is missing in cerebellar motor learning (16). Despite the apparently contradictory results, no direct evidence for the decrease or increase of synaptic AMPA receptors (AMPARs) has been shown in physiological motor learning. To elucidate in vivo neuronal substrates for motor learning in wildtype mouse, we examined individual PF-PC synapses using quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL) (17) combined with morphometric EM analysis after adaptation of ho...

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Quantum Dots for Detection, Identification and Tracking of Single Biomolecules in Tissue and Cells

Loukanov¹,

Emin²

2012

Intelligent Nanomaterials

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The study of basic cell ultrastructure and intracellular physiological functions has been greatly aided by detection and identification of single macromolecules. Since the current in situ labeling methods for directly correlative (light and electron) microscopy observations have a number of substantial limitations, the semiconductor nanocrystals quantum dots gain distinguished with long-term imaging and high photostability. The quantum dots (Qdots) have quickly filled in the role, being found to be superior to traditional organic dyes on several counts. It has been estimated that Qdots are 20 times brighter and 100 times more stable than traditional fluorescent reporters. Nowadays, a wide variety of quantum dots conjugated to secondary antibodies suitable for multiple labeling have become commercially available. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. By applying Qdots with different size and color light we might achieve multiple labeling of proteins. However, the use of particles with different size is problematic for high-resolution imaging, semi-quantitative measurement of epitope numbers, or when epitope density is high. Recently we report new electron microscopy method for immunolabeling, where two approaches are performed to distinguish yet unattainable spatial

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Number and Density of AMPA Receptors in Individual Synapses in the Rat Cerebellum as Revealed by SDS-Digested Freeze-Fracture Replica Labeling

Cited by 160 publications

References 55 publications

Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors

Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors

Distinct cerebellar engrams in short-term and long-term motor learning

Quantum Dots for Detection, Identification and Tracking of Single Biomolecules in Tissue and Cells

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