SDS-digested Freeze-fracture Replica Labeling Research Articles

Nanoscale Phosphoinositide Distribution on Cell Membranes of Mouse Cerebellar Neurons.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons.SIGNIFICANCE STATEMENT In this study, we established an electron microscopic method to visualize and analyze the quantitative distribution pattern of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) on cell membranes using cryo-fixed brain tissues and SDS-digested freeze-fracture replica labeling. PI(4,5)P2 interacts with various ion channels and receptors to regulate membrane signaling but its nanoscale distribution and association with these proteins remain elusive. This method revealed PI(4,5)P2 clusters preferentially accumulated in specific membrane compartments and its distinct associations with CaV2.1, GIRK3, and mGluR1α in the mouse cerebellum. These results demonstrate usefulness of the method for gaining insights into the physiological functions of PI(4,5)P2.

Alteration in the Synaptic and Extrasynaptic Organization of AMPA Receptors in the Hippocampus of P301S Tau Transgenic Mice.

Tau pathology is a hallmark of Alzheimer's disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD.

Age-Dependent Shift of AMPA Receptors From Synapses to Intracellular Compartments in Alzheimer's Disease: Immunocytochemical Analysis of the CA1 Hippocampal Region in APP/PS1 Transgenic Mouse Model.

Synapse loss occurs early in Alzheimer’s disease (AD) patients and animal models. Alterations at synaptic level are a major morphological correlate of the memory deficits and related symptoms of AD. Given the predominant roles of synaptic AMPA receptors (AMPARs) in excitatory synaptic transmission in the brain, changes in their dynamic regulation are also implicated in the pathophysiology of AD. Here, we used immunolocalization techniques to analyze the expression and subcellular distribution of AMPARs in the hippocampal region of APP/PS1 mouse model of AD. Immunoblots and histoblots revealed that the total amount of AMPARs and their regional expression pattern in the hippocampus was similar in APP/PS1 mice and in age-matched wild type mice. At the ultrastructural level, two synapse populations were examined using SDS-digested freeze-fracture replica labeling in the stratum radiatum in mice: (i) on spines of CA1 pyramidal cells; and (ii) on randomly found dendritic shafts of CA1 interneurons. While 1- and 6-months-old APP/PS1 mice exhibited no change, we observed a significant reduction at 12 months in AMPAR density at synapses in both pyramidal cells and interneurons, compared to wild-type. This reduction of AMPARs in dendritic spines was accompanied by a significant increase in AMPAR subunit proteins identified in intracellular compartments. Our data demonstrate an age-dependent reduction of synaptic AMPARs in APP/PS1 mice, which may contribute to impaired learning and memory at later stages of AD.

Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain.

In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.

SK2 Channels Associate With mGlu1α Receptors and CaV2.1 Channels in Purkinje Cells

The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs.

Functional deficiency of MHC class I enhances LTP and abolishes LTD in the nucleus accumbens of mice.

Major histocompatibility complex class I (MHCI) molecules were recently identified as novel regulators of synaptic plasticity. These molecules are expressed in various brain areas, especially in regions undergoing activity-dependent synaptic plasticity, but their role in the nucleus accumbens (NAc) is unknown. In this study, we investigated the effects of genetic disruption of MHCI function, through deletion of β2-microblobulin, which causes lack of cell surface expression of MHCI. First, we confirmed that MHCI molecules are expressed in the NAc core in wild-type mice. Second, we performed electrophysiological recordings with NAc core slices from wild-type and β2-microglobulin knock-out mice lacking cell surface expression of MHCI. We found that low frequency stimulation induced long-term depression in wild-type but not knock-out mice, whereas high frequency stimulation induced long-term potentiation in both genotypes, with a larger magnitude in knock-out mice. Furthermore, we demonstrated that knock-out mice showed more persistent behavioral sensitization to cocaine, which is a NAc-related behavior. Using this model, we analyzed the density of total AMPA receptors and their subunits GluR1 and GluR2 in the NAc core, by SDS-digested freeze-fracture replica labeling. After repeated cocaine exposure, the density of GluR1 was increased, but there was no change in total AMPA receptors and GluR2 levels in wild-type mice. In contrast, following repeated cocaine exposure, increased densities of total AMPA receptors, GluR1 and GluR2 were observed in knock-out mice. These results indicate that functional deficiency of MHCI enhances synaptic potentiation, induced by electrical and pharmacological stimulation.

Quantitative Localization of Cav2.1 (P/Q-Type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering with Calcium-Activated Potassium Channels

P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Ca(v)2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Ca(v)2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Ca(v)2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Ca(v)2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Ca(v)2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ∼40 nm. Calcium nanodomain created by the opening of Ca(v)2.1 channels likely activates the two channels that limit the extent of depolarization.

Kv4.2 potassium channels segregate to extrasynaptic domains and influence intrasynaptic NMDA receptor NR2B subunit expression

Neurons of the intercalated cell clusters (ITCs) represent an important relay site for information flow within amygdala nuclei. These neurons receive mainly glutamatergic inputs from the basolateral amygdala at their dendritic domains and provide feed-forward inhibition to the central nucleus. Voltage-gated potassium channels type-4.2 (Kv4.2) are main players in dendritic signal processing and integration providing a key component of the A currents. In this study, the subcellular localization and distribution of the Kv4.2 was studied in ITC neurons by means of light- and electron microscopy, and compared to other types of central principal neurons. Several ultrastructural immunolocalization techniques were applied including pre-embedding techniques and, most importantly, SDS-digested freeze-fracture replica labeling. We found Kv4.2 densely expressed in somato-dendritic domains of ITC neurons where they show a differential distribution pattern as revealed by nearest neighbor analysis. Comparing ITC neurons with hippocampal pyramidal and cerebellar granule cells, a cell type- and domain-dependent organization in Kv4.2 distribution was observed. Kv4.2 subunits were localized to extrasynaptic sites where they were found to influence intrasynaptic NMDA receptor subunit expression. In samples of Kv4.2 knockout mice, the frequency of NR1-positive synapses containing the NR2B subunit was significantly increased. This indicates a strong, yet indirect effect of Kv4.2 on the synaptic content of NMDA receptor subtypes, and a likely role in synaptic plasticity at ITC neurons.

A unique distribution pattern of the Kv4.2 channels on hippocampal CA1 pyramidal cells is revealed by freeze-fracture replica labeling

Event Abstract Back to Event A unique distribution pattern of the Kv4.2 channels on hippocampal CA1 pyramidal cells is revealed by freeze-fracture replica labeling K. M. Kerti1, A. Lőrincz1 and Z. Nusser1* 1 Hungarian Academy of Sciences, Institute of Experimental Medicine, Hungary A-current flowing through Kv4.2 voltage-gated K+ channels plays a critical role in controlling pyramidal cell (PC) excitability. Electrophysiological recordings have demonstrated a 6-fold increase in A-type K+ current density along the somato-dendritic axis of hippocampal CA1 PCs. However, the underlying channel distribution and densities remained unknown. Here we examined the subcellular distribution and densities of Kv4.2 subunits in CA1 PC somata and 11 distinct dendritic compartments using a highly sensitive immunogold method (SDS-digested freeze-fracture replica labeling). Only a moderate (only 50%) increase in Kv4.2 immunogold density was observed along the proximo-distal axis of apical dendrites in the stratum radiatum with a slight decrease in stratum lacunosum-moleculare. This was characteristic to all dendritic compartments, including main apical dendrites, oblique dendrites and dendritic spines. In addition to the somato-dendritic localization, the Kv4.2 subunit was also found in axon terminals at a 4-fold lower density. Our results demonstrate a novel distribution pattern of the Kv4.2 subunit along the axo-somato-dendritic surface of CA1 PCs and suggest that the strong increase in the A-current density cannot be solely explained by a corresponding increase in channel number. Keywords: Molecular and cellular neurobiology, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Molecular and cellular neurobiology Citation: Kerti KM, Lőrincz A and Nusser Z (2011). A unique distribution pattern of the Kv4.2 channels on hippocampal CA1 pyramidal cells is revealed by freeze-fracture replica labeling. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00153 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. Z. Nusser, Hungarian Academy of Sciences, Institute of Experimental Medicine, Budapest, Hungary, nusser@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers K. M Kerti A. Lőrincz Z. Nusser Google K. M Kerti A. Lőrincz Z. Nusser Google Scholar K. M Kerti A. Lőrincz Z. Nusser PubMed K. M Kerti A. Lőrincz Z. Nusser Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Differential postsynaptic compartments in the laterocapsular division of the central nucleus of amygdala for afferents from the parabrachial nucleus and the basolateral nucleus in the rat

Neurons in the laterocapsular division of the central nucleus of the amygdala (CeC), which is known as the "nociceptive amygdala," receive glutamatergic inputs from the parabrachial nucleus (PB) and the basolateral nucleus of amygdala (BLA), which convey nociceptive information from the dorsal horn of the spinal cord and polymodal information from the thalamus and cortex, respectively. Here, we examined the ultrastructural properties of PB- and BLA-CeC synapses identified with EGFP-expressing lentivirus in rats. In addition, the density of synaptic AMPA receptors (AMPARs) on CeC neurons was studied by using highly sensitive SDS-digested freeze-fracture replica labeling (SDS-FRL). Afferents from the PB made asymmetrical synapses mainly on dendritic shafts (88%), whereas those from the BLA were on dendritic spines (81%). PB-CeC synapses in dendritic shafts were significantly larger (median 0.072 μm(2)) than BLA-CeC synapses in spines (median 0.058 μm(2); P = 0.02). The dendritic shafts that made synapses with PB fibers were also significantly larger than those that made synapses with BLA fibers, indicating that the PB fibers make synapses on more proximal parts of dendrites than the BLA fibers. SDS-FRL revealed that almost all excitatory postsynaptic sites have AMPARs in the CeC. The density of AMPAR-specific gold particles in individual synapses was significantly higher in spine synapses (median 510 particles/μm(2)) than in shaft synapses (median 427 particles/μm(2); P = 0.01). These results suggest that distinct synaptic impacts from PB- and BLA-CeC pathways contribute to the integration of nociceptive and polymodal information in the CeC.

Two distinct pools of large-conductance calcium-activated potassium channels in the somatic plasma membrane of central principal neurons

Although nerve cell membranes are often assumed to be uniform with respect to electrical properties, there is increasing evidence for compartmentalization into subdomains with heterogeneous impacts on the overall cell function. Such microdomains are characterized by specific sets of proteins determining their functional properties. Recently, clustering of large-conductance calcium-activated potassium (BKCa) channels was shown at sites of subsurface membrane cisterns in cerebellar Purkinje cells (PC), where they likely participate in building a subcellular signaling unit, the 'PLasmERosome'. By applying SDS-digested freeze-fracture replica labeling (SDS-FRL) and postembedding immunogold electron microscopy, we have now studied the spatial organization of somatic BKCa channels in neocortical layer 5 pyramidal neurons, principal neurons of the central and basolateral amygdaloid nuclei, hippocampal pyramidal neurons and dentate gyrus (DG) granule cells to establish whether there is a common organizational principle in the distribution of BKCa channels in central principal neurons. In all cell types analyzed, somatic BKCa channels were found to be non-homogenously distributed in the plasma membrane, forming two pools of channels with one pool consisting of clustered channels and the other of scattered channels in the extrasynaptic membrane. Quantitative analysis by means of SDS-FRL revealed that about two-thirds of BKCa channels belong to the scattered pool and about one-third to the clustered pool in principal cell somata. Overall densities of channels in both pools differed in the different cell types analyzed, although being considerably lower compared to cerebellar PC. Postembedding immunogold labeling revealed association of clustered channels with subsurface membrane cisterns and confirmed extrasynaptic localization of scattered channels. This study indicates a common organizational principle for somatic BKCa channels in central principal neurons with the formation of a clustered and a scattered pool of channels, and a cell-type specific density of this channel type.

Molecular identity of dendritic voltage-gated sodium channels in CA1 pyramidal cells

Event Abstract Back to Event Molecular identity of dendritic voltage-gated sodium channels in CA1 pyramidal cells Andrea Lorincz1* and Zoltán Nusser1 1 Hungarian Academy of Sciences, Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Hungary High density of voltage-gated sodium (Nav) channels in axon initial segments (AIS) of pyramidal cells (PC) ensures low threshold for the initiation of action potential (AP). Nav channels-mediated currents were found in the somato-dendritic domain of PCs, where they contribute to dendritic spikes and backpropagating APs. However, the molecular identity of somato-dendritic Nav channels is unknown, most likely owing to the limited sensitivity of the applied methods. Here we used immunofluorescent reactions and a highly sensitive electron microscopic SDS-digested freeze-fracture replica-labeling (SDS-FRL) technique to identify the Nav subunit content of the axo-somato-dendritic plasma membranes of CA1 pyramidal cells (PCs). Modified immunofluorescent reactions with a pan-Nav antibody demonstrated the presence of Nav channels in the axo-somato-dendritic compartments of CA1 PCs, but the method failed to detect Nav1.1, Nav1.2 or Nav1.6 subunits on the somato-dendritic surface of CA1 PCs. Using SDS-FRL, high densities of gold particles for the Nav1.6 subunit were detected in Nodes of Ranvier and AISs of CA1 PCs. We found a graded increase in the density of gold particles along the proximo-distal axis of AISs and the gold particles consistently avoided GABAergic axo-axonic synapses. Somatic and dendritic plasma membranes also contained gold particles for the Nav1.6 subunit at low, but significant densities. However, we could not detect gold particles in dendritic spines. Quantitative analysis revealed that AISs contained ~40-fold higher density of gold particles than somatic and proximal dendritic plasma membranes. A distance dependent decrease in the density of gold particles was also observed along the proximo-distal axis of the dendrites. Our results demonstrate for the first time the somato-dendritic presence of the Nav1.6 subunit and reveal quantitative differences in the amount of the Nav1.6 subunit between different subcellular compartments of CA1 PCs. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Cellular neuroscience Citation: Lorincz A and Nusser Z (2010). Molecular identity of dendritic voltage-gated sodium channels in CA1 pyramidal cells. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00078 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Apr 2010; Published Online: 21 Apr 2010. * Correspondence: Andrea Lorincz, Hungarian Academy of Sciences, Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Budapest, Hungary, lorincz@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Andrea Lorincz Zoltán Nusser Google Andrea Lorincz Zoltán Nusser Google Scholar Andrea Lorincz Zoltán Nusser PubMed Andrea Lorincz Zoltán Nusser Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Input-Specific Intrasynaptic Arrangements of Ionotropic Glutamate Receptors and Their Impact on Postsynaptic Responses

To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.

Large‐conductance calcium‐activated potassium channels in purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains

Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells.

BK channels in Purkinje cell plasma membranes are concentrated in plasmerosomes at sites of hypolemmal cisternae

Calcium-activated potassium channels have been shown to be critically involved in neuronal function but an elucidation of their detailed roles awaits identification of the subcellular domains and microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the big-conductance calcium-activated potassium channels (called BK, KCa1.1 or Slo1) in the somato-dendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane and localized to specific subcellular domains. At distal dendritic compartments, BK channels were scattered over the plasma membrane of shafts and spines, but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, the other pool was clustered in plasma membrane domains overlying subsurface membrane cisterns, also called hypolemmal cisternae. These subcompartments of the endoplasmic reticulum likely represent calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells.

Application of SDS-digested Freeze-fracture Replica Labeling for Molecular Localization in Neural Tissues

Application of SDS-digested Freeze-fracture Replica Labeling for Molecular Localization in Neural Tissues

Formation of aquaporin-4 arrays is inhibited by palmitoylation of N-terminal cysteine residues

Tetramers of the mammalian water channel aquaporin-4 (AQP4) assemble into square arrays and mediate bidirectional water transport across the blood–brain interface. The aqp4 gene expresses two splicing isoforms. Only the shorter AQP4M23 isoform assembles into square arrays, while the longer AQP4M1 isoform interferes with array formation, presumably due to the additional 22 N-terminal residues. To understand why the N-terminus of AQP4M1 interferes with array formation, we constructed a series of N-terminal deletion mutants and examined their ability to form square arrays in Chinese hamster ovary (CHO) cells using SDS-digested freeze fracture replica labeling. Mutants with deletions of less than seventeen N-terminal residues did not form square arrays and showed dispersed immunogold labels against AQP4 molecules, whereas more deletions led to the formation of square arrays labeled with immunogolds. Furthermore, mutagenic substitution of the two cysteine residues at the position 13 and 17 in the N-terminus of AQP4M1 also resulted in the square array formation. Biochemical analysis and metabolic labeling of transfected CHO cells revealed that the two N-terminal cysteines of AQP4M1 are palmitoylated. These results suggest that palmitoylation of the N-terminal cysteines is the reason for the inability of AQP4M1 to form square arrays.

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

The number of AMPA receptor (AMPAR) is the major determinant of synaptic strength at glutamatergic synapses, but little is known about the absolute number and density of AMPARs in individual synapses. Using SDS-digested freeze-fracture replica labeling, which has high detection efficiency comparable with electrophysiological noise analysis for functional AMPAR, we analyzed three kinds of excitatory synapses in the molecular layer of the adult rat cerebellum. In parallel fiber (PF)-Purkinje cell (PC) synapses, we found large variability in the number (38.1 +/- 34.4 particles per synapse, mean +/- SD; range, 2-178 particles per synapse) and density (437 +/- 277 particles/microm2; range, 48-1210 particles/microm2) of immunogold-labeled AMPARs. Two-dimensional view and high sensitivity of this method revealed irregular-shaped small AMPAR clusters within synapses. Climbing fiber (CF)-PC synapses had higher number of AMPAR labeling (68.6 +/- 34.5 particles per synapse) than PF-PC and PF-interneuron synapses (36.8 +/- 14.4 particles per synapse). Furthermore, AMPAR density at CF-PC and PF-interneuron synapses was approximately five times higher and more uniform than that at PF-PC synapses. These results suggest input- and target-dependent regulation of AMPAR-mediated synaptic strength.

A New Method to Visualize the Helicobacter pylori-associated Lewisb-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

Freeze-fracture replica labeling has become a versatile tool to visualize both membrane components and other cell structures using SDS-replica cleaning before specific immunogold labeling of proteins or lipids. We report here for the first time the adoption and optimization of the method to studies of bacterial envelopes, as applied to structural analysis of the distribution of the unique BabA-adhesin of the gastric pathogen Helicobacter pylori. BabA is important for bacterial adherence to the human epithelial cell lining of the stomach. The adhesin was found to be distributed all over the bacterial cell surfaces. Our results suggest that the SDS-replica labeling allows assessment of protein localization to distinct cell compartments and analysis of co-localization with neighboring membrane structures.

SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application.

Recently, we have developed a quick-freezing/freeze-fracture replica labeling technique, sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL), to study the two-dimensional distribution of cytochemical labeling on the membrane surface and the relationship of this distribution to images of freeze-fracture replicas created by platinum shadowing. In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon shadowing, are treated with SDS. The detergent dissolves unfractured areas of the cell membranes, with the release of the cytoplasmic contents. The cytoplasmic and exoplasmic membrane surfaces can be then labeled cytochemically. Integral membrane proteins, revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody. In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes. In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy.