Freeze-fracture Replicas Research Articles

Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons.

It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the rodent hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse.

Definition of phosphatidylinositol 4,5-bisphosphate distribution by freeze-fracture replica labeling.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a phospholipid essential for plasma membrane functions, but its two-dimensional distribution is not clear. Here, we compared the result of sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)/eisosome, especially in the curved MCC/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1δ-PH. PtdIns(4,5)P2 remaining after acute ATP depletion and in the stationary phase, 30.0% and 56.6% of the control level, respectively, was not detectable by fluorescence biosensor, whereas the label intensity by SDS-FRL reflected the PtdIns(4,5)P2 amount. In PC12 cells, PtdIns(4,5)P2 was observed in a punctate pattern in the formaldehyde-fixed plasma membrane, whereas it was distributed randomly by SDS-FRL and showed clustering after formaldehyde fixation. The results indicate that the distribution of PtdIns(4,5)P2 can be defined most reliably by SDS-FRL of quick-frozen cells.

Selective disruption of synaptic NMDA receptors of the hippocampal trisynaptic circuit in Aβ pathology

Synaptic dysfunction is an early feature in Alzheimer’s disease (AD) pathogenesis and a major morphological correlate of memory deficits. Given the main synaptic location of N-methyl-D-aspartate receptors (NMDARs), their dysregulation has been implicated in these pathological effects. Here, to detect possible alterations in the expression and synaptic localisation of the GluN1 subunit in the brain of amyloidogenic APP/PS1 mice, we employed histoblot and SDS-digested freeze-fracture replica labelling (SDS-FRL) techniques. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus in a layer-dependent manner, in the cortex and the caudate putamen of APP/PS1 transgenic mice at 12 months of age but was unaltered at 1 and 6 months. Using quantitative SDS-FRL, we unravelled the molecular organisation of GluN1 in seven excitatory synapse populations at a high spatial resolution in the CA1 and CA3 fields and the DG of the hippocampus in 12-month-old APP/PS1 mice. In the CA1 field, the labelling density for GluN1 in the excitatory synapses established on spines and interneurons, was significantly reduced in APP/PS1 mice compared to age-matched wild-type mice in the stratum lacunosum-moleculare but unaltered in the stratum radiatum. In the CA3 field, synaptic GluN1 was reduced in mossy fibre-CA3 pyramidal cell synapses but unaltered in the A/C-CA3 pyramidal cell synapses. In the DG, the density of GluN1 in granule cell-perforant pathway synapses was reduced in APP/PS1 mice. Altogether, our findings provide evidence of specific alterations of synaptic GluN1 in the trisynaptic circuit of the hippocampus in Aβ pathology. This differential vulnerability in the disruption of NMDARs may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit and cognitive impairment characteristic of APP/PS1 mice.

Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse

The coupling between Ca2+ channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse. Biophysical analysis of transmission by paired recording and intracellular pipette perfusion revealed that the effects of exogenous Ca2+ chelators decreased during development, despite constant reliance of release on P/Q-type Ca2+ channels. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission.

Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis.

Toxoplasma gondii is a highly prevalent obligate apicomplexan parasite that is important in clinical and veterinary medicine. It is known that glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn), especially their expression levels and flip-flops between cytoplasmic and exoplasmic leaflets, in the membrane of T. gondii play important roles in efficient growth in host mammalian cells, but their distributions have still not been determined because of technical difficulties in studying intracellular lipid distribution at the nanometer level. In this study, we developed an electron microscopy method that enabled us to determine the distributions of PtdSer and PtdEtn in individual leaflets of cellular membranes by using quick-freeze freeze-fracture replica labeling. Our findings show that PtdSer and PtdEtn are asymmetrically distributed, with substantial amounts localized at the luminal leaflet of the inner membrane complex (IMC), which comprises flattened vesicles located just underneath the plasma membrane (see Figs. 2B and 7). We also found that PtdSer was absent in the cytoplasmic leaflet of the inner IMC membrane, but was present in considerable amounts in the cytoplasmic leaflet of the middle IMC membrane, suggesting a barrier-like mechanism preventing the diffusion of PtdSer in the cytoplasmic leaflets of the two membranes. In addition, the expression levels of both PtdSer and PtdEtn in the luminal leaflet of the IMC membrane in the highly virulent RH strain were higher than those in the less virulent PLK strain. We also found that the amount of glycolipid GM3, a lipid raft component, was higher in the RH strain than in the PLK strain. These results suggest a correlation between lipid raft maintenance, virulence, and the expression levels of PtdSer and PtdEtn in T. gondii.

Subcellular distribution of membrane lipids revealed by freeze-fracture electron microscopy.

Cell membranes are composed of a large variety of lipids and proteins. While the localization and function of membrane proteins have been extensively investigated, the distribution of membrane lipids, especially in the non-cytoplasmic leaflet of organelle membranes, remains largely unknown. Fluorescent biosensors have been widely used to study membrane lipid distribution; however, they have some limitations. By utilizing the quick-freezing and freeze-fracture replica labeling electron microscopy technique, we can uncover the precise distribution of membrane lipids within cells and assess the function of lipid-transporting proteins. In this review, I summarize recent progress in analyzing intracellular lipid distribution by utilizing this method.

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.

Protein Networks Associated with Native Metabotropic Glutamate 1 Receptors (mGlu1) in the Mouse Cerebellum.

The metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses.

Tight-junction strand networks and tightness of the epithelial barrier.

Tight junctions (TJs) are cell-cell junction structures critical for controlling paracellular permeability. On freeze-fracture replica electron microscopy, they appear as a continuous network of fibrils (TJ strands). TJ strands function as zippers that create a physical barrier against paracellular diffusion of molecules. The morphology of the TJ strand network varies greatly between tissues, and in recent years, studies have highlighted the mechanisms regulating the morphology of TJ strand networks and on their relevance to barrier function. In this review, we discuss evidence regarding the components of the TJ strand and the mechanisms for creating the TJ strand network. Furthermore, we discuss and hypothesize how its morphology contributes to the establishment of the epithelial barrier.

Effect of Polyphenols on the Ice-Nucleation Activity of Ultrafine Bubbles.

Ultrafine bubbles (UFBs) in water provide a large amount of gas and a large gas-liquid interfacial area, and can release energy through their collapse. Such features may promote ice nucleation. Here, we examined the nucleation of ice in solutions containing polyphenols and UFBs. To reduce the likelihood of nucleation occurring on the container walls over that in previous studies, we used a much larger sample volume of 1 mL. In our experiments, UFBs (when present) had a number concentration of 108 mL-1. We quantified changes to the nucleation activity by examining the shift in the cumulative freezing (nucleation) probability distribution. Compared to pure water, this freezing curve shifts approximately 0.6 °C higher with the UFBs. Then, to the water, we added three polyphenols (tannic acid TA, tea catechin TC, and oligonol OLG), chosen because they had been reported to reduce the ice-nucleation activity of heterogeneous ice nuclei (e.g., AgI). We found experimentally that, without UFBs, all polyphenols instead shift the pure-water freezing curve to a higher temperature. Then, when UFBs are added, the additional temperature shift in the freezing curve is slightly higher for OLG, essentially unchanged for TA, and slightly lower for TC. To help to explain these differences, we examined the UFB size distributions using dynamic light scattering and freeze-fractured replicas with transmission electron microscopy, finding that OLG and TC alter the UFBs, but that TA does not.

Distribution of Glycolipids in the Plasma Membrane Monitored by Specific Probes in Combination with Sodium Dodecyl Sulfate-Digested Freeze-Fracture Replica Labeling (SDS-FRL).

Glycolipids are mainly distributed in the outer leaflet of the plasma membrane and are involved in cellular signaling by modulating the activity of cell surface receptor proteins. Glycolipids themselves also work as cell surface receptors of bacterial toxins. Anti-glycolipid antibodies are associated with various pathological conditions. The cellular distribution of glycolipids has been studied using specific toxins or antibodies. However, these proteins are multivalent and thus potentially induce the artificial aggregation of glycolipids. Since chemical fixative such as paraformaldehyde does not fix glycolipids, an alternative methodology is required to localize glycolipids with multivalent probes. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) physically fixes glycolipids on the cast after quick freezing. Thus, SDS-FRL provides the opportunity to observe the natural distribution of glycolipids using multivalent probes. Here, we describe the application of SDS-FRL on the cell surface distribution of phosphatidylglucoside.

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.

On-grid labeling method for freeze-fracture replicas.

Sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) is an electron microscopic (EM) method that can define the two-dimensional distribution of membrane proteins and lipids in a quantitative manner. Despite its unsurpassed merit, SDS-FRL has been adopted in a limited number of labs, probably because it requires a laborious labeling process as well as equipment and technique for freeze-fracture. Here, we present a method that reduces the manual labor significantly by mounting freeze-fracture replicas on EM grids prior to labeling. This was made possible by the discovery that freeze-fracture replicas invariably adhere to the carbon-coated formvar membrane with their platinum-carbon side, ensuring that the membrane molecules retained in replicas are accessible to labeling solutions. The replicas mounted on EM grids can be stored dry until labeling, checked by light microscopy before labeling and labeled in the same manner as tissue sections. This on-grid method will make SDS-FRL easier to access for many researchers.

Nanoscale alterations in GABAB receptors and GIRK channel organization on the hippocampus of APP/PS1 mice

Alzheimer’s disease (AD) is characterized by a reorganization of brain activity determining network hyperexcitability and loss of synaptic plasticity. Precisely, a dysfunction in metabotropic GABAB receptor signalling through G protein-gated inwardly rectifying K+ (GIRK or Kir3) channels on the hippocampus has been postulated. Thus, we determined the impact of amyloid-β (Aβ) pathology in GIRK channel density, subcellular distribution, and its association with GABAB receptors in hippocampal CA1 pyramidal neurons from the APP/PS1 mouse model using quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL) and proximity ligation in situ assay (P-LISA). In wild type mice, single SDS-FRL detection revealed a similar dendritic gradient for GIRK1 and GIRK2 in CA1 pyramidal cells, with higher densities in spines, and GIRK3 showed a lower and uniform distribution. Double SDS-FRL showed a co-clustering of GIRK2 and GIRK1 in post- and presynaptic compartments, but not for GIRK2 and GIRK3. Likewise, double GABAB1 and GIRK2 SDS-FRL detection displayed a high degree of co-clustering in nanodomains (40–50 nm) mostly in spines and axon terminals. In APP/PS1 mice, the density of GIRK2 and GIRK1, but not for GIRK3, was significantly reduced along the neuronal surface of CA1 pyramidal cells and in axon terminals contacting them. Importantly, GABAB1 and GIRK2 co-clustering was not present in APP/PS1 mice. Similarly, P-LISA experiments revealed a significant reduction in GABAB1 and GIRK2 interaction on the hippocampus of this animal model. Overall, our results provide compelling evidence showing a significant reduction on the cell surface density of pre- and postsynaptic GIRK1 and GIRK2, but not GIRK3, and a decline in GABAB receptors and GIRK2 channels co-clustering in hippocampal pyramidal neurons from APP/PS1 mice, thus suggesting that a disruption in the GABAB receptor–GIRK channel membrane assembly causes dysregulation in the GABAB signalling via GIRK channels in this AD animal model.

Different modes of synaptic and extrasynaptic NMDA receptor alteration in the hippocampus of P301S tau transgenic mice.

N-methyl-d-aspartate receptors (NMDARs) are pivotal players in the synaptic transmission and synaptic plasticity underlying learning and memory. Accordingly, dysfunction of NMDARs has been implicated in the pathophysiology of Alzheimer disease (AD). Here, we used histoblot and sodium dodecylsulphate-digested freeze-fracture replica labelling (SDS-FRL) techniques to investigate the expression and subcellular localisation of GluN1, the obligatory subunit of NMDARs, in the hippocampus of P301S mice. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered at 3 months. Using the SDS-FRL technique, excitatory synapses and extrasynaptic sites on spines of pyramidal cells and interneuron dendrites were analysed throughout all dendritic layers in the CA1 field. Our ultrastructural approach revealed a high density of GluN1 in synaptic sites and a substantially lower density at extrasynaptic sites. Labelling density for GluN1 in excitatory synapses established on spines was significantly reduced in P301S mice, compared with age-matched wild-type mice, in the stratum oriens (so), stratum radiatum (sr) and stratum lacunosum-moleculare (slm). Density for synaptic GluN1 on interneuron dendrites was significantly reduced in P301S mice in the so and sr but unaltered in the slm. Labelling density for GluN1 at extrasynaptic sites showed no significant differences in pyramidal cells, and only increased density in the interneuron dendrites of the sr. This differential alteration of synaptic versus extrasynaptic NMDARs supports the notion that the progressive accumulation of phospho-tau is associated with changes in NMDARs, in the absence of amyloid-β pathology, and may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit.

Gold In-and-Out: A Toolkit for Analyzing Subcellular Distribution of Immunogold-Labeled Membrane Proteins in Freeze-Fracture Replica Images.

Integral membrane proteins such as ion channels, transporters, and receptors shape cell activity and mediate cell-to-cell communication in the brain. The distribution, quantity, and clustering arrangement of those proteins contribute to the physiological properties of the cell; therefore, precise quantification of their state can be used to gain insight into cellular function. Using a highly sensitive immunoelectron microscopy technique called sodium dodecyl sulfate-digested freeze-fracture replica immunogold labeling (SDS-FRL), multiple membrane proteins can be tagged with different sizes of immunogold particles at once and visualized two-dimensionally. For quantification, gold particles in the images must be annotated, and then different mathematical and statistical methods must be applied to characterize the distribution states of proteins of interest. To perform such analyses in a user-friendly manner, we developed a program with a simple graphical user interface called Gold In-and-Out (GIO), which integrates several classical and novel analysis methods for immunogold labeled replicas into one self-contained package. GIO takes an input of particle coordinates, then allows users to implement analysis methods such as nearest neighbor distance (NND) and particle clustering. The program not only performs the selected analysis but also automatically compares the results of the real distribution to a random distribution of the same number of particles on the membrane region of interest. In addition to classical approaches for analyzing protein distribution, GIO includes new tools to analyze the positional bias of a target protein relative to a morphological landmark such as dendritic spines, and can also be applied for synaptic protein analysis. Gold Rippler provides a normalized metric of particle density that is resistant to differences in labeling efficiency among samples, while Gold Star is useful for quantifying distances between a protein and landmark. This package aims to help standardize analysis methods for subcellular and synaptic protein localization with a user-friendly interface while increasing the efficiency of these time-consuming analyses.

The Number and Distinct Clustering Patterns of Voltage-Gated Calcium Channels in Nerve Terminals.

Upon the arrival of action potentials at nerve terminals, neurotransmitters are released from synaptic vesicles (SVs) by exocytosis. CaV2.1, 2.2, and 2.3 are the major subunits of the voltage-gated calcium channel (VGCC) responsible for increasing intraterminal calcium levels and triggering SV exocytosis in the central nervous system (CNS) synapses. The two-dimensional analysis of CaV2 distributions using sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) has revealed their numbers, densities, and nanoscale clustering patterns in individual presynaptic active zones. The variation in these properties affects the coupling of VGCCs with calcium sensors on SVs, synaptic efficacy, and temporal precision of transmission. In this study, we summarize how the morphological parameters of CaV2 distribution obtained using SDS-FRL differ depending on the different types of synapses and could correspond to functional properties in synaptic transmission.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization.

Simple SummaryGap junctions are intercellular channels that provide the means for direct transport of small molecules, ions, and water between connected cells. With these functions, gap junctions are essential for the maintenance of astrocytic homeostasis and of particular importance in the context of pathophysiological disbalances. These include the hyperosmolar hyperglycemic syndrome or the pathology after brain trauma. We demonstrate that short-term hyperosmolarity reduces intercellular communication via gap junctions. These functional changes coincide with the transformation of gap junction ultrastructure as evidenced by freeze-fracture replica immunolabeling and transmission electron microscopy. The hyperosmolarity-induced immediate changes in the ultrastructural assembly of connexons, the protein constituents of gap junction channels, have not been described in astrocytes before and are revealing the coherence of structure and function in gap junctions. Phosphorylation of Connexin 43, the main gap junction protein in astrocytes, at amino acid 368 (Serine) might link the two.In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Fear Memory Retrieval Is Associated With a Reduction in AMPA Receptor Density at Thalamic to Amygdala Intercalated Cell Synapses.

The amygdala plays a crucial role in attaching emotional significance to environmental cues. Its intercalated cell masses (ITC) are tight clusters of GABAergic neurons, which are distributed around the basolateral amygdala complex. Distinct ITC clusters are involved in the acquisition and extinction of conditioned fear responses. Previously, we have shown that fear memory retrieval reduces the AMPA/NMDA ratio at thalamic afferents to ITC neurons within the dorsal medio-paracapsular cluster. Here, we investigate the molecular mechanisms underlying the fear-mediated reduction in the AMPA/NMDA ratio at these synapses and, in particular, whether specific changes in the synaptic density of AMPA receptors underlie the observed change. To this aim, we used a detergent-digested freeze-fracture replica immunolabeling technique (FRIL) approach that enables to visualize the spatial distribution of intrasynaptic AMPA receptors at high resolution. AMPA receptors were detected using an antibody raised against an epitope common to all AMPA subunits. To visualize thalamic inputs, we virally transduced the posterior thalamic complex with Channelrhodopsin 2-YFP, which is anterogradely transported along axons. Using face-matched replica, we confirmed that the postsynaptic elements were ITC neurons due to their prominent expression of μ-opioid receptors. With this approach, we show that, following auditory fear conditioning in mice, the formation and retrieval of fear memory is linked to a significant reduction in the density of AMPA receptors, particularly at spine synapses formed by inputs of the posterior intralaminar thalamic and medial geniculate nuclei onto identified ITC neurons. Our study is one of the few that has directly linked the regulation of AMPA receptor trafficking to memory processes in identified neuronal networks, by showing that fear-memory induced reduction in AMPA/NMDA ratio at thalamic-ITC synapses is associated with a reduced postsynaptic AMPA receptor density.

A deep learning approach to identifying immunogold particles in electron microscopy images

Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community.