Postreplication labeling of E‐leaflet molecules: Membrane immunoglobulins localized in sectioned, labeled replicas examined by TEM and HVEM

Dinchuk, Joseph; Johnson, Timothy J. A.; Rash, John E.

doi:10.1002/jemt.1060070102

Cited by 35 publications

(42 citation statements)

References 36 publications

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“…This washing procedure leaves a thin film of lipid and protein molecules adhering to the highly absorptive platinum-carbon replica (Fujimoto 1995), plus additional molecules that are bound to the molecules that are directly adsorbed to the replica (Fujimoto 1995;Rash and Yasumura 1999). SDS-washed replicas were rinsed in "blocking buffer" [10% heatinactivated goat serum and 1.5% fish gelatin in Sorenson's phosphate buffer, pH 7.4 (Dinchuk et al 1987)] and labeled for 60 -220 min using rabbit polyclonal antibodies to Cx36 [Ab298 (Rash et al 2000;Pereda et al 2003a); from Dr. James Nagy, University of Manitoba, Winnipeg, Canada] with or without monoclonal antibodies to glutamate receptor subunit NR1 (Ab55-6308; BD Biosciences PharMingen, San Diego, CA). Replicas of goldfish Mauthner cells were labeled using polyclonal antibody Ab298 and monoclonal antibodies to NR1 (both as described above).…”

Section: Freeze-fracture Replica Immunolabelingmentioning

confidence: 99%

The extent and strength of electrical coupling between inferior olivary neurons is heterogeneous

Hoge

Davidson

Yasumura

et al. 2011

Journal of Neurophysiology

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Gap junctions constitute the only form of synaptic communication between neurons in the inferior olive (IO), which gives rise to the climbing fibers innervating the cerebellar cortex. Although its exact functional role remains undetermined, electrical coupling was shown to be necessary for the transient formation of functional compartments of IO neurons and to underlie the precise timing of climbing fibers required for cerebellar learning. So far, most functional considerations assume the existence of a network of permanently and homogeneously coupled IO neurons. Contrasting this notion, our results indicate that coupling within the IO is highly variable. By combining tracer-coupling analysis and paired electrophysiological recordings, we found that individual IO neurons could be coupled to a highly variable number of neighboring neurons. Furthermore, a given neuron could be coupled at remarkably different strengths with each of its partners. Freeze-fracture analysis of IO glomeruli revealed the close proximity of glutamatergic postsynaptic densities to connexin 36-containing gap junctions, at distances comparable to separations between chemical transmitting domains and gap junctions in goldfish mixed contacts, where electrical coupling was shown to be modulated by the activity of glutamatergic synapses. On the basis of structural and molecular similarities with goldfish mixed synapses, we speculate that, rather than being hardwired, variations in coupling could result from glomerulus-specific long-term modulation of gap junctions. This striking heterogeneity of coupling might act to finely influence the synchronization of IO neurons, adding an unexpected degree of complexity to olivary networks.

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Section: Freeze-fracture Replica Immunolabelingmentioning

confidence: 99%

The extent and strength of electrical coupling between inferior olivary neurons is heterogeneous

Hoge

Davidson

Yasumura

et al. 2011

Journal of Neurophysiology

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“…29 h at 48°-50°C in 2.5% SDS detergent (Fujimoto, 1995), and sites of non-specific binding to the highly adsorbent platinum and carbon layers of the replica (Dinchuk et al, 1987;Rash et al, 1989;Fujimoto, 1995) were blocked in a solution of 1.5% fish gelatin digest plus 10% heat inactivated goat serum (both from Sigma-Aldrich, St. Louis, MO) (Dinchuk et al, 1987) in Sørensen's phosphate buffer. Replicas were immunogold labeled using one, two or three of eight different affinity-purified antibodies to four CNS connexins (Cx26, Cx32, Cx36 and Cx47; see Table 1, antibodies used at 10 μg/ml).…”

Section: Freeze-fracture Replica Immunogold Labeling (Fril)mentioning

confidence: 99%

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

et al. 2007

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Locus coeruleus neurons are strongly coupled during early postnatal development, and it has been proposed that these neurons are linked by extraordinarily abundant gap junctions consisting of connexin32 and connexin26, and that those same connexins abundantly link neurons to astrocytes. Based on the controversial nature of those claims, immunofluorescence imaging and freeze-fracture replica immunogold labeling were used to re-investigate the abundance and connexin composition of neuronal and glial gap junctions in developing and adult rat and mouse locus coeruleus. In early postnatal development, connexin36 and Cx43 immunofluorescent puncta were densely distributed in the locus coeruleus, whereas connexin32 and connexin26 were not detected. By freeze-fracture replica immunogold labeling, connexin36 was found in ultrastructurally-defined neuronal gap junctions, whereas connexin32 and connexin26 were not detected in neurons and only rarely detected in glia. In 28-day postnatal (adult) rat locus coeruleus, immunofluorescence labeling for connexin26 was always co-localized with the glial gap junction marker connexin43; connexin32 was associated with the oligodendrocyte marker CNPase; and connexin36 was never co-localized with connexin26, Cx32 or connexin43. Ultrastructurally, connexin36 was localized to gap junctions between neurons, whereas connexin32 was detected only in oligodendrocyte gap junctions; and Cx26 was found only rarely in astrocyte junctions but abundantly in pia mater. Thus, in developing and adult locus coeruleus, neuronal gap junctions contain connexin36 but do not contain detectable connexin32 or connexin26, suggesting that the locus coeruleus has the same cell-type specificity of connexin expression as observed ultrastructurally in other regions of the central nervous system. Moreover, in both developing and adult locus coeruleus, no evidence was found for gap junctions or connexins linking neurons with astrocytes or oligodendrocytes, indicating that neurons in this nucleus are not linked to the pan-glial syncytium by connexin32-or connexin26-containing gap junctions or by abundant free connexons composed of those connexins. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2008 July 29. Published in final edited form as:Neuroscience. In the developing mammalian central nervous system (CNS), electrical coupling between neurons is well documented by electrophysiological and dye-coupling approaches (Peinado et al., 1993;Fulton, 1995;Montoro and Yuste, 2004;Bruzzone and...

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“…Before immunolabeling, nonspecific binding sites of the SDS-washed replicas were blocked by preincubation in labeling blocking buffer (LBB) consisting of 10% heat-inactivated goat serum, 0.5% teleost gelatin, and 0.05% sodium azide in 150 mM SPB (Dinchuk et al, 1987;Rash et al, 1990). Antibodies at a stock concentration of 1 mg/ml were diluted 1:100 in LBB, either using single antibodies or mixed antibodies of two species.…”

Section: Sources Of Tissuementioning

confidence: 99%

Connexin32-Containing Gap Junctions in Schwann Cells at the Internodal Zone of Partial Myelin Compaction and in Schmidt–Lanterman Incisures

Meier¹,

Dermietzel²,

Davidson³

et al. 2004

J. Neurosci.

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In vertebrate peripheral nerves, the insulating myelin sheath is formed by Schwann cells, which generate flattened membrane processes that spiral around axons and form compact myelin by extrusion of cytoplasm and adhesion of apposed intracellular and extracellular membrane surfaces. Cytoplasm remains within the innermost and outermost tongues, in the paranodal loops bordering nodes of Ranvier and in Schmidt-Lanterman incisures. By immunocytochemistry, connexin32 (Cx32) protein has been demonstrated at paranodal loops and Schmidt-Lanterman incisures, and it is widely assumed that gap junctions are present in these locations, thereby providing a direct radial route for transport of ions and metabolites between cytoplasmic myelin layers. This study used freeze-fracture replica immunogold labeling to detect Cx32 in ultrastructurally defined gap junctions in Schmidt-Lanterman incisures, as well as in a novel location, between the outer two layers of internodal myelin, approximately every micrometer along the entire length of myelin, at the zone between compact myelin and noncompact myelin. Thus, these gap junctions link the partially compacted second layer of myelin to the noncompact outer tongue. Although these gap junctions are unusually small (average, 11 connexon channels), their relative abundance and regular distribution along the zone that is structurally intermediate between compact and noncompact myelin demonstrates the existence of multiple sites for unidirectional or bidirectional transport of water, ions, and small molecules between these two distinct cytoplasmic compartments, possibly to regulate or facilitate myelin compaction or to maintain the transition zone between noncompact and compact myelin.

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Postreplication labeling of E‐leaflet molecules: Membrane immunoglobulins localized in sectioned, labeled replicas examined by TEM and HVEM

Cited by 35 publications

References 36 publications

The extent and strength of electrical coupling between inferior olivary neurons is heterogeneous

The extent and strength of electrical coupling between inferior olivary neurons is heterogeneous

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

Connexin32-Containing Gap Junctions in Schwann Cells at the Internodal Zone of Partial Myelin Compaction and in Schmidt–Lanterman Incisures

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