Modality-Specific Impairment of Hippocampal CA1 Neurons of Alzheimer's Disease Model Mice.

Impairment of episodic memory, a class of memory for spatiotemporal context of an event, is an early symptom of Alzheimer's disease. Both spatial and temporal information are encoded and represented in the hippocampal neurons, but how these representations are impaired under amyloid ß (Aß) pathology remains elusive. We performed chronic imaging of the hippocampus in awake male amyloid precursor protein (App) knock-in mice behaving in a virtual reality environment to simultaneously monitor spatiotemporal representations and the progression of Aß depositions. We found that temporal representation is preserved, whereas spatial representation is significantly impaired in the App knock-in mice. This is because of the overall reduction of active place cells, but not time cells, and compensatory hyperactivation of remaining place cells near Aß aggregates. These results indicate the differential impact of Aß aggregates on two major modalities of episodic memory, suggesting different mechanisms for forming and maintaining these two representations in the hippocampus.

Reduced axonal localization of a Caps2 splice variant impairs axonal release of BDNF and causes autistic-like behavior in mice.

Ca(2)(+)-dependent activator protein for secretion 2 (CAPS2 or CADPS2) potently promotes the release of brain-derived neurotrophic factor (BDNF). A rare splicing form of CAPS2 with deletion of exon3 (dex3) was identified to be overrepresented in some patients with autism. Here, we generated Caps2-dex3 mice and verified a severe impairment in axonal Caps2-dex3 localization, contributing to a reduction in BDNF release from axons. In addition, circuit connectivity, measured by spine and interneuron density, was diminished globally. The collective effect of reduced axonal BDNF release during development was a striking and selective repertoire of deficits in social- and anxiety-related behaviors. Together, these findings represent a unique mouse model of a molecular mechanism linking BDNF-mediated coordination of brain development to autism-related behaviors and patient genotype.

CAPS1 deficiency perturbs dense-core vesicle trafficking and Golgi structure and reduces presynaptic release probability in the mouse brain.

Ca(2+)-dependent activator protein for secretion 1 (CAPS1) plays a regulatory role in the dense-core vesicle (DCV) exocytosis pathway, but its functions at the cellular and synaptic levels in the brain are essentially unknown because of neonatal death soon after birth in Caps1 knock-out mice. To clarify the functions of the protein in the brain, we generated two conditional knock-out (cKO) mouse lines: 1) one lacking Caps1 in the forebrain; and 2) the other lacking Caps1 in the cerebellum. Both cKO mouse lines were born normally and grew to adulthood, although they showed subcellular and synaptic abnormalities. Forebrain-specific Caps1 cKO mice showed reduced immunoreactivity for the DCV marker secretogranin II (SgII) and the trans-Golgi network (TGN) marker syntaxin 6, a reduced number of presynaptic DCVs, and dilated trans-Golgi cisternae in the CA3 region. Cerebellum-specific Caps1 cKO mice had decreased immunoreactivity for SgII and brain-derived neurotrophic factor (BDNF) along the climbing fibers. At climbing fiber-Purkinje cell synapses, the number of DCVs was markedly lower and the number of synaptic vesicles was also reduced. Correspondingly, the mean amplitude of EPSCs was decreased, whereas paired-pulse depression was significantly increased. Our results suggest that loss of CAPS1 disrupts the TGN-DCV pathway, which possibly impairs synaptic transmission by reducing the presynaptic release probability.

Virtual reality-based real-time imaging reveals abnormal cortical dynamics during behavioral transitions in a mouse model of autism.

Functional connectivity (FC) can provide insight into cortical circuit dysfunction in neuropsychiatric disorders. However, dynamic changes in FC related to locomotion with sensory feedback remain to be elucidated. To investigate FC dynamics in locomoting mice, we develop mesoscopic Ca2+ imaging with a virtual reality (VR) environment. We find rapid reorganization of cortical FC in response to changing behavioral states. By using machine learning classification, behavioral states are accurately decoded. We then use our VR-based imaging system to study cortical FC in a mouse model of autism and find that locomotion states are associated with altered FC dynamics. Furthermore, we identify FC patterns involving the motor area as the most distinguishing features of the autism mice from wild-type mice during behavioral transitions, which might correlate with motor clumsiness in individuals with autism. Our VR-based real-time imaging system provides crucial information to understand FC dynamics linked to behavioral abnormality of neuropsychiatric disorders.

Interaction of calcium-dependent activator protein for secretion 1 (CAPS1) with the class II ADP-ribosylation factor small GTPases is required for dense-core vesicle trafficking in the trans-Golgi network.

Ca(2+)-dependent activator protein for secretion (CAPS) regulates exocytosis of catecholamine- or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites, such as nerve terminals. However, large amounts of CAPS protein are localized in the cell soma, and the role of somal CAPS protein remains unclear. The present study shows that somal CAPS1 plays an important role in DCV trafficking in the trans-Golgi network. The anti-CAPS1 antibody appeared to pull down membrane fractions, including many Golgi-associated proteins, such as ADP-ribosylation factor (ARF) small GTPases. Biochemical analyses of the protein-protein interaction showed that CAPS1 interacted specifically with the class II ARF4/ARF5, but not with other classes of ARFs, via the pleckstrin homology domain in a GDP-bound ARF form-specific manner. The pleckstrin homology domain of CAPS1 showed high affinity for the Golgi membrane, thereby recruiting ARF4/ARF5 to the Golgi complex. Knockdown of either CAPS1 or ARF4/ARF5 expression caused accumulation of chromogranin, a DCV marker protein, in the Golgi, thereby reducing its DCV secretion. In addition, the overexpression of CAPS1 binding-deficient ARF5 mutants induced aberrant chromogranin accumulation in the Golgi and consequently reduced its DCV secretion. These findings implicate a functional role for CAPS1 protein in the soma, a major subcellular localization site of CAPS1 in many cell types, in regulating DCV trafficking in the trans-Golgi network; this activity occurs via protein-protein interaction with ARF4/ARF5 in a GDP-dependent manner.

Autistic-like phenotypes in Cadps2-knockout mice and aberrant CADPS2 splicing in autistic patients.

Autism, characterized by profound impairment in social interactions and communicative skills, is the most common neurodevelopmental disorder, and its underlying molecular mechanisms remain unknown. Ca(2+)-dependent activator protein for secretion 2 (CADPS2; also known as CAPS2) mediates the exocytosis of dense-core vesicles, and the human CADPS2 is located within the autism susceptibility locus 1 on chromosome 7q. Here we show that Cadps2-knockout mice not only have impaired brain-derived neurotrophic factor release but also show autistic-like cellular and behavioral phenotypes. Moreover, we found an aberrant alternatively spliced CADPS2 mRNA that lacks exon 3 in some autistic patients. Exon 3 was shown to encode the dynactin 1-binding domain and affect axonal CADPS2 protein distribution. Our results suggest that a disturbance in CADPS2-mediated neurotrophin release contributes to autism susceptibility.

Expression of the IP3R1 promoter-driven nls-lacZ transgene in Purkinje cell parasagittal arrays of developing mouse cerebellum.

The cerebellar Purkinje cell monolayer is organized into heterogeneous Purkinje cell compartments that have different molecular compositions. Here we describe a transgenic mouse line, 1NM13, that shows heterogeneous transgene expression in parasagittal Purkinje cell arrays. The transgene consists of a nuclear localization signal (nls) fused to the beta-galactosidase (lacZ) composite gene driven by the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) gene promoter. IP(3)R1-nls-lacZ transgene expression was detected at a single Purkinje cell level over the surface of a whole-mount X-gal-stained cerebellum because of nuclear accumulation of the nls-lacZ activity. Developing cerebella of 1NM13 mice showed stripe-like X-gal staining patterns of parasagittal Purkinje cell subsets. The X-gal stripe pattern was likely determined by an intrinsic property as early as E15 and showed increasing complexity with cerebellar development. The X-gal stripe pattern was reminiscent of, but not identical to, the stripe pattern of zebrin II immunoreactivity. We designated the symmetrical X-gal-positive (transgene-positive, Tg(+)) Purkinje cell stripes about the midline as vermal Tg1(+), Tg2(a, b)(+) and Tg3(a, b)(+) stripes and hemispheric Tg4(a, b)(+), Tg5(a, b)(+), Tg6(a, b, c)(+), and Tg7(a, b)(+) stripes, where a, b, and c indicate substripes. We also assigned three parafloccular substripes Tg8(a, b, c)(+). The boundaries of X-gal stripes at P5 were consistent with raphes in the Purkinje cell layer through which granule cells migrate, suggesting a possible association of the X-gal stripes with raphe formation. Our results indicate that 1NM13 is a good mouse model with a reproducible and clear marker for the compartmentalization of Purkinje cell arrays.

Distinct Mechanisms of Over-Representation of Landmarks and Rewards in the Hippocampus.

In the hippocampus, locations associated with salient features are represented by a disproportionately large number of neurons, but the cellular and molecular mechanisms underlying this over-representation remain elusive. Using longitudinal calcium imaging in mice learning to navigate in virtual reality, we find that the over-representation of reward and landmark locations are mediated by persistent and separable subsets of neurons, with distinct time courses of emergence and differing underlying molecular mechanisms. Strikingly, we find that in mice lacking Shank2, an autism spectrum disorder (ASD)-linked gene encoding an excitatory postsynaptic scaffold protein, the learning-induced over-representation of landmarks was absent whereas the over-representation of rewards was substantially increased, as was goal-directed behavior. These findings demonstrate that multiple hippocampal coding processes for unique types of salient features are distinguished by a Shank2-dependent mechanism and suggest that abnormally distorted hippocampal salience mapping may underlie cognitive and behavioral abnormalities in a subset of ASDs.

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 learning/control. The possible involvement of the CAPS2 gene in the cerebellar deficits of autistic patients is discussed.

Cerebellar development transcriptome database (CDT-DB): profiling of spatio-temporal gene expression during the postnatal development of mouse cerebellum.

A large amount of genetic information is devoted to brain development and functioning. The neural circuit of the mouse cerebellum develops through a series of cellular and morphological events (including neuronal proliferation and migration, axogenesis, dendritogenesis, synaptogenesis and myelination) all within three weeks of birth. All of these events are controlled by specific gene groups, whose temporal and spatial expression profiles must be encoded in the genome. To understand the genetic basis underlying cerebellar circuit development, we analyzed gene expression (transcriptome) during the developmental stages on a genome-wide basis. Spatio-temporal gene expression data were collected using in situ hybridization for spatial (cellular and regional) resolution and fluorescence differential display, GeneChip, microarray and RT-PCR for temporal (developmental time series) resolution, and were annotated using Gene Ontology (controlled terminology for genes and gene products) and anatomical context (cerebellar cell types and circuit structures). The annotated experimental data were integrated into a knowledge resource database, the Cerebellar Development Transcriptome Database (CDT-DB http://www.cdtdb.brain.riken.jp), with seamless links to the relevant information at various bioinformatics database websites. The CDT-DB not only provides a unique informatics tool for mining both spatial and temporal pattern information on gene expression in developing mouse brains, but also opens up opportunities to elucidate the transcriptome for cerebellar development.

Differential distributions of the Ca2+ -dependent activator protein for secretion family proteins (CAPS2 and CAPS1) in the mouse brain.

The Ca(2+)-dependent activator protein for secretion (CAPS/Cadps) family consists of two members, CAPS1 and CAPS2, and plays an important role in secretory granule exocytosis. It has been shown that CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), from parallel fibers of cerebellar granule cells. Although both CAPS proteins are expressed predominantly in the brain, their cellular and regional distributions in the brain are largely unknown. In this study we analyzed the immunohistochemical distributions of the CAPS family proteins in the mouse brain. In most areas of the embryonic nervous system CAPS1 and CAPS2 proteins were complementarily expressed. In the postnatal brain, CAPS1 was widespread at different levels. On the other hand, CAPS2 was localized to distinct cell types and fibers of various brain regions, including the olfactory bulb, cerebrum, hippocampal formation, thalamus, mesencephalic tegmentum, cerebellum, medulla, and spinal cord, except for some regions that overlapped with CAPS1. These CAPS2 cellular distribution patterns had the marked feature of coinciding with those of BDNF in various brain regions. Immunolabels for CAPS2 were also colocalized with those for some proteins related to exocytosis (VAMP and SNAP-25) and endocytosis (Dynamin I) in the cell soma and processes of the mesencephalic tegmentum and cerebellum, suggesting that these proteins might be involved in the dynamics of CAPS2-associated vesicles, although their colocalization on vesicles remains elusive. These results demonstrate that the CAPS family proteins are involved in the secretion of different secretory substances in developing and postnatal brains, and that CAPS2 is probably involved in BDNF secretion in many brain areas.

Inhibiting the Activity of CA1 Hippocampal Neurons Prevents the Recall of Contextual Fear Memory in Inducible ArchT Transgenic Mice.

The optogenetic manipulation of light-activated ion-channels/pumps (i.e., opsins) can reversibly activate or suppress neuronal activity with precise temporal control. Therefore, optogenetic techniques hold great potential to establish causal relationships between specific neuronal circuits and their function in freely moving animals. Due to the critical role of the hippocampal CA1 region in memory function, we explored the possibility of targeting an inhibitory opsin, ArchT, to CA1 pyramidal neurons in mice. We established a transgenic mouse line in which tetracycline trans-activator induces ArchT expression. By crossing this line with a CaMKIIα-tTA transgenic line, the delivery of light via an implanted optrode inhibits the activity of excitatory CA1 neurons. We found that light delivery to the hippocampus inhibited the recall of a contextual fear memory. Our results demonstrate that this optogenetic mouse line can be used to investigate the neuronal circuits underlying behavior.

Serodiagnosis of canine Babesia gibsoni infection by enzyme-linked immunosorbent assay with recombinant P50 expressed in Escherichia coli.

The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni-infected, Babesia canis-infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni. When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni. This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coil is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.

Axonal localization of Ca2+-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum.

Ca2+-dependent activator protein for secretion 2 (CAPS2) is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic) of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.

Autistic-like behavioral phenotypes in a mouse model with copy number variation of the CAPS2/CADPS2 gene.

Ca²âº-dependent activator protein for secretion 2 (CAPS2 or CADPS2) facilitates secretion and trafficking of dense-core vesicles. Recent genome-wide association studies of autism have identified several microdeletions due to copy number variation (CNV) in one of the chromosome 7q31.32 alleles on which the locus for CAPS2 is located in autistic patients. To evaluate the biological significance of reducing CAPS2 copy number, we analyzed CAPS2 heterozygous mice. Our present findings suggest that adequate levels of CAPS2 protein are critical for normal brain development and behavior, and that allelic changes due to CNV may contribute to autistic symptoms in combination with deficits in other autism-associated genes.

Calcium-dependent activator protein for secretion 2 interacts with the class II ARF small GTPases and regulates dense-core vesicle trafficking.

The Ca(2+) -dependent activator protein for secretion (CAPS) family consists of two members (CAPS1 and CAPS2) and regulates the exocytosis of catecholamine-containing or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites such as nerve terminals. A large fraction of CAPS1, however, is localized in the cell soma, and we have recently shown the possible involvement of somal CAPS1 in DCV trafficking in the trans-Golgi network. CAPS1 and CAPS2 are differentially expressed in various regions of the mouse brain but exhibit similar expression patterns in other tissues, such as the spleen. Thus, in the present study we analyzed whether CAPS2 displays similar subcellular localization and functional roles in the cell soma as CAPS1. We found that somal CAPS2 is associated with the Golgi membrane, and mediates binding and recruitment of the GDP-bound form of ARF4 and ARF5 (members of the membrane-trafficking small GTPase family) to the Golgi membrane. CAPS2 knockdown and overexpression of CAPS2-binding-deficient ARF4/ARF5 both induced accumulation of the DCV resident protein chromogranin A around the Golgi apparatus. CAPS2 knockout mice have dilated trans-Golgi structures when viewed by electron microscopy. These results for CAPS2 strongly support our idea that the CAPS family proteins exert dual roles in DCV trafficking, mediating trafficking at both the secretion site for exocytosis and at the Golgi complex for biogenesis.

Suitable solutions for reconstituting the ultrasound contrast agent "Levovist" used in contrast echocardiogram: in vitro and in vivo evaluation of the influence of osmotic pressure.

PURPOSE: To evaluate the influence of the osmotic pressure of solutions used for reconstituting the ultrasound contrast agent "Levovist" on the degree of video intensity of the enhancement and video intensity decay in contrast echocardiogram, we used 6 solutions with different osmotic pressures in both vitro and in vivo experiments. MATERIALS AND METHODS: In the in vitro experiments, Levovist was reconstituted with 6 kinds of solutions with different osmotic pressures (Lactate Ringer's solution, 2.5%, 5%, 7.5%, and 20% glucose and distilled water) and peak video intensities and video intensity decay were measured. In the in vivo experiments, contrast echocardiography was performed in 7 adult volunteers, following the intravenous injections of Levovist, previously reconstituted with one of 2 solutions (5% glucose or distilled water). RESULTS: In vitro, at peak time, Levovist reconstituted with either Lactate Ringer's solution, 2.5% glucose, 5% glucose, or distilled water had good peak video intensities. At 30 s after peak time, Levovist reconstituted with Lactate Ringer's solution had greater enhancement and less decay than the other 5 solutions (P<0.001). In vivo, at 180 heart beats after peak time, the video intensity decay with 5% glucose was greater than that with distilled water (150+/-13 dB, 123+/-25 dB, respectively, P<0.05). CONCLUSION: In this study, among various (2.5-20%) glucose solutions, the stability of the microbubbles differed, depending on the degree of osmotic pressure of the respective solutions; 5% glucose was the best. However, overall, the most suitable solution for reconstituting Levovist, was Lactate Ringer's solution. These findings could lead to effective strategies for better contrast echocardiography using Levovist by changing the current solution of choice to Lactate Ringer's solution or 5% glucose.

Doppler imaging predicts cardiac events in chronic pulmonary thromboembolism.

PURPOSE: We evaluated whether right ventricular (RV) diastolic dysfunction assessed by pulsed tissue Doppler imaging (TDI) predicts cardiac events in patients with chronic pulmonary thromboembolism (CPTE). MATERIALS AND METHODS: In 63 consecutive patients with CPTE, early diastolic myocardial velocity (Ea) at the tricuspid annulus by TDI and early diastolic tricuspid inflow (E) by conventional pulsed Doppler were obtained, and E/Ea was calculated as an indicator of RV diastolic dysfunction. Brain natriuretic peptide (BNP) and other echo parameters were also obtained. A cardiac event (rehospitalization caused by congestive heart failure or cardiac death) was the study endpoint. Incidence of cardiac events was determined over a 374+/-451 day follow-up period. RESULTS: In the follow-up period twelve patients had cardiac events. We divided patients into group A with cardiac events and group B without events. E/Ea was significantly increased in group A as compared with group B (8.3+/-4.1 vs. 5.7+/-2.6, p<0.01). BNP was higher in group A than group B (221+/-191 vs. 121+/-140 mg/dl, p<0.05), and in addition E/Ea was significantly positively correlated with BNP (r=0.48, p<0.001). A logistic regression model for predicting cardiac events was constructed and E/Ea was associated with an increased incidence of cardiac events (relative risk=1.33, 95% CI 1.00-1.75). CONCLUSION: Elevated values of E/Ea obtained by TDI may predict cardiac events in patients with CPTE. BNP may also be a significant predictor.

Opalin, a transmembrane sialylglycoprotein located in the central nervous system myelin paranodal loop membrane.

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1-30), a transmembrane domain (residues 31-53), and a long C-terminal intracellular domain (residues 54-143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.