Postembedding Immunogold Electron Microscopy Research Articles

Analytical Post-Embedding Immunogold-Electron Microscopy with Direct Gold-Labelled Monoclonal Primary Antibodies against RIBEYE A- and B-Domain Suggests a Refined Model of Synaptic Ribbon Assembly.

Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold-electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.

Gut region-specific TNFR expression: TNFR2 is more affected than TNFR1 in duodenal myenteric ganglia of diabetic rats.

Cytokines are essential in autoimmune inflammatory processes that accompany type 1 diabetes. Tumor necrosis factor alpha plays a key role among others in modulating enteric neuroinflammation, however, it has a dual role in cell degeneration or survival depending on different TNFRs. In general, TNFR1 is believed to trigger apoptosis, while TNFR2 promotes cell regeneration. The importance of the neuronal microenvironment has been recently highlighted in gut region-specific diabetic enteric neuropathy, however, the expression and alterations of different TNFRs in the gastrointestinal tract has not been reported. To investigate the TNFR1 and TNFR2 expression in myenteric ganglia and their environment in different intestinal segments of diabetic rats. Ten weeks after the onset of hyperglycemia, gut segments were taken from the duodenum, ileum and colon of streptozotocin-induced (60 mg/body weight kg i.p.) diabetic (n = 17), insulin-treated diabetic (n = 15) and sex- and age-matched control (n = 15) rats. Myenteric plexus whole-mount preparations were prepared from different gut regions for TNFR1/HuCD or TNFR2/HuCD double-labeling fluorescent immunohistochemistry. TNFR1 and TNFR2 expression was evaluated by post-embedding immunogold electron microscopy on ultrathin sections of myenteric ganglia. TNFRs levels were measured by enzyme-linked immun-osorbent assay in muscle/myenteric plexus-containing (MUSCLE-MP) tissue homogenates from different gut segments and experimental conditions. A distinct region-dependent TNFRs expression was detected in controls. The density of TNFR1-labeling gold particles was lowest, while TNFR2 density was highest in duodenal ganglia and a decreased TNFRs expression from proximal to distal segments was observed in MUSCLE-MP homogenates. In diabetics, the TNFR2 density was only significantly altered in the duodenum with decrease in the ganglia (0.32 ± 0.02 vs 0.45 ± 0.04, P < 0.05), while no significant changes in TNFR1 density was observed. In diabetic MUSCLE-MP homogenates, both TNFRs levels significantly decreased in the duodenum (TNFR1: 4.06 ± 0.65 vs 20.32 ± 3.1, P < 0.001; TNFR2: 11.72 ± 0.39 vs 15.91 ± 1.04, P < 0.01), which markedly influenced the TNFR2/TNFR1 proportion in both the ganglia and their muscular environment. Insulin treatment had controversial effects on TNFR expression. Our findings show diabetes-related region-dependent changes in TNFR expression and suggest that TNFR2 is more affected than TNFR1 in myenteric ganglia in the duodenum of type 1 diabetic rats.

Diabetes-related intestinal region-specific thickening of ganglionic basement membrane and regionally decreased matrix metalloproteinase 9 expression in myenteric ganglia.

BACKGROUNDThe importance of the neuronal microenvironment has been recently highlighted in gut region-specific diabetic enteric neuropathy. Regionally distinct thickening of endothelial basement membrane (BM) of intestinal capillaries supplying the myenteric ganglia coincide with neuronal damage in different intestinal segments. Accelerated synthesis of matrix molecules and reduced degradation of matrix components may also contribute to the imbalance of extracellular matrix dynamics resulting in BM thickening. Among the matrix degrading proteinases, matrix metalloproteinase 9 (MMP9) and its tissue inhibitor (TIMP1) are essential in regulating extracellular matrix remodelling.AIMTo evaluate the intestinal segment-specific effects of diabetes and insulin replacement on ganglionic BM thickness, MMP9 and TIMP1 expression.METHODSTen weeks after the onset of hyperglycaemia gut segments were taken from the duodenum and ileum of streptozotocin-induced diabetic, insulin-treated diabetic and sex- and age-matched control rats. The thickness of BM surrounding myenteric ganglia was measured by electron microscopic morphometry. Whole-mount preparations of myenteric plexus were prepared from the different gut regions for MMP9/TIMP1 double-labelling fluorescent immunohistochemistry. Post-embedding immunogold electron microscopy was applied on ultrathin sections to evaluate the MMP9 and TIMP1 expression in myenteric ganglia and their microenvironment from different gut segments and conditions. The MMP9 and TIMP1 messenger ribonucleic acid (mRNA) level was measured by quantitative polymerase chain reaction.RESULTSTen weeks after the onset of hyperglycaemia, the ganglionic BM was significantly thickened in the diabetic ileum, while it remained intact in the duodenum. The immediate insulin treatment prevented the diabetes-related thickening of the BM surrounding the ileal myenteric ganglia. Quantification of particle density showed an increasing tendency for MMP9 and a decreasing tendency for TIMP1 from the proximal to the distal small intestine under control conditions. In the diabetic ileum, the number of MMP9-indicating gold particles decreased in myenteric ganglia, endothelial cells of capillaries and intestinal smooth muscle cells, however, it remained unchanged in all duodenal compartments. The MMP9/TIMP1 ratio was also decreased in ileal ganglia only. However, a marked segment-specific induction was revealed in MMP9 and TIMP1 at the mRNA levels.CONCLUSIONThese findings support that the regional decrease in MMP9 expression in myenteric ganglia and their microenvironment may contribute to extracellular matrix accumulation, resulting in a region-specific thickening of ganglionic BM.

Upregulation of the lactate transporter monocarboxylate transporter 1 at the blood-brain barrier in a rat model of attention-deficit/hyperactivity disorder suggests hyperactivity could be a form of self-treatment

The energy deficit hypothesis of attention-deficit/hyperactivity disorder (ADHD) suggests that low lactate production by brain astrocytes causes the symptoms of the disorder. Astrocytes are the main producers of lactate in the brain; however, skeletal muscles can produce the most lactate in the body. The lactate production by skeletal muscles increases with physical activity, as does the expression of the lactate transporter monocarboxylate transporter 1 (MCT1) at the blood-brain barrier (BBB). We hypothesise that children with ADHD, by being hyperactive, increase lactate production by skeletal muscles and transport it into the brain to compensate for low supply by astrocytes. The aim of this study was to explore whether the level of MCT1 is altered in the brain in an animal model of ADHD. The MCT1 expression was quantified on hippocampal brain sections from the best available rat model of ADHD, i.e., the spontaneously hypertensive rat (SHR) (n = 12), and the relevant control, the Wistar Kyoto rat (WKY) (n = 12), by the use of quantitative immunofluorescence laser scanning microscopy and postembedding immunogold electron microscopy. The results revealed significantly higher levels of hippocampal MCT1 immunoreactivity in SHR compared to WKY, particularly at the BBB. These results indicate that lactate flux through MCT1 between the body and the brain could be upregulated in children with ADHD. This study adds to previous research suggesting hyperactivity may be beneficial in ADHD; Children with ADHD possibly display a hyperactive behaviour in order to raise skeletal muscle lactate production, MCT1 expression and flux over the BBB to supply the brain with lactate.

Changes in synaptic AMPA receptor concentration and composition in chronic temporal lobe epilepsy

Excitotoxicity caused by excessive stimulation of glutamate receptors, resulting in pathologically increased Ca2+-concentrations, is a decisive factor in neurodegenerative diseases. We investigated long-term changes in synaptic contents of AMPA receptor subunits that play important roles in calcium regulation in chronic epilepsy. Such plastic changes may be either adaptive or detrimental. We used a kainic acid (KA)-based rat model of chronic temporal lobe epilepsy (TLE). Using hippocampal synaptosomes, we found significant reductions in the concentration of the AMPA receptor subunits GluA1 and GluA2, and the NMDA receptor subunit NR2B. The relative size of GluA1 and GluA2 reductions were almost identical, at 28% and 27%, respectively. In order to determine whether the synaptic reduction of the AMPA receptor subunits actually reflected the pool of receptors present along the postsynaptic density (PSD), as opposed to cytoplasmic or extrasynaptic pools, we performed postembedding immunogold electron microscopy (EM) of GluA1 and GluA2 in Schaffer collateral synapses in the hippocampal CA1 area. We found significant reductions, at 32% and 52% of GluA1 and GluA2 subunits, respectively, along the PSD, indicating that these synapses undergo lasting changes in glutamatergic neurotransmission during chronic TLE. When compared to the overall concentration and composition of AMPA receptors expressed in the brain, there was a relative increase in GluA2-lacking AMPA receptor subunits following chronic epilepsy. These changes in synaptic AMPA receptor subunits may possibly contribute to further aggravate the excitotoxic vulnerability of the neurons as well as have significant implications for hippocampal cognitive functions.

The calcium sensor synaptotagmin 1 is expressed and regulated in hippocampal postsynaptic spines.

Synaptotagmin 1 is a presynaptic calcium sensor, regulating SNARE-mediated vesicle exocytosis of transmitter. Increasing evidence indicate roles of SNARE proteins in postsynaptic glutamate receptor trafficking. However, a possible postsynaptic expression of synaptotagmin 1 has not been demonstrated previously. Here, we used postembedding immunogold electron microscopy to determine the subsynaptic localization of synaptotagmin 1 in rat hippocampal CA1 Schaffer collateral synapses. We report for the first time that synaptotagmin 1 is present in rat hippocampal postsynaptic spines, both on cytoplasmic vesicles and at the postsynaptic density. We further investigated whether postsynaptic synaptotagmin 1 is regulated during synaptic plasticity. In a rat model of chronic temporal lobe epilepsy, we found that presynaptic and postsynaptic concentrations of the protein are reduced compared to control animals. This downregulation may possibly be an adaptive measure to decrease both presynaptic and postsynaptic calcium sensitivity in excitotoxic conditions.

DISTINCT ULTRASTRUCTURAL CHARACTERISTICS OF CAPILLARY BASAL LAMINA IN HUMAN AND MOUSE BRAINS: RELEVANCE TO MURINE MODELS OF NEUROVASCULAR DISEASE

Recent studies suggest that capillary dysfunction may be a source of neurodegeneration, stroke and cognitive decline (J Cereb Blood Flow Metab, 2016, 36:302; Neurobiol Aging, 2017, 50:107). Most neurovascular studies used mice to model human diseases. The morphologic changes in the capillary wall are less-well characterized in humans, especially in early or presymptomatic phases and in early onset diseases. Here we describe major differences in ultrastructure of capillary basal lamina (CBL) in humans (normal and diseased, <50 years) compared with mouse (young to middle age). Human autopsy brains without or with neurodegeneration, including Parkinson's disease, frontotemporal degeneration and amyotrophic lateral sclerosis, and mouse models of tauopathy were evaluated. Brain tissues were fixed in formaldehyde and/or glutaraldehyde and processed for electron microscopy (EM) or post-embedding immunogold EM. Antibodies to types I, III and IV collagen and to fibronectin were used. Mouse CBL is a thin and uniformly amorphous structure that is labeled with anti-type IV collagen in both normal mice and models of neurodegenerative disease at all ages. In contrast, the CBL in normal and diseased human brains has the amorphous BL, but also banded collagens. In addition, the BL frequently shows segmental splitting between endothelial and astrocytic sides of the BL. The split creates an expanded space that contains banded fibrils of type I and III collagen, but not type IV collagen. The expansion and splitting of BL increase with age and in diseased brains. The CBL in diseased human brains often extends into the pericapillary space. Anti-fibronectin co-localizes with collagens. These ultrastructural features are rarely observed in mice, but are detected in the youngest human brains studied. Human CBL has age- and disease-related alternations including split BL and accumulation of banded type I and III collagen fibrils associated with fibronectin. This BL “fibrosis” is rarely observed in mice brains. If these structural BL alterations are associated with capillary dysfunction, the lack of these changes in mice warrants caution about use of mouse models for neurovascular studies.

Maternally involved galanin neurons in the preoptic area of the rat.

Recent selective stimulation and ablation of galanin neurons in the preoptic area of the hypothalamus established their critical role in control of maternal behaviors. Here, we identified a group of galanin neurons in the anterior commissural nucleus (ACN), and a distinct group in the medial preoptic area (MPA). Galanin neurons in ACN but not the MPA co-expressed oxytocin. We used immunodetection of phosphorylated STAT5 (pSTAT5), involved in prolactin receptor signal transduction, to evaluate the effects of suckling-induced prolactin release and found that 76% of galanin cells in ACN, but only 12% in MPA were prolactin responsive. Nerve terminals containing tuberoinfundibular peptide 39 (TIP39), a neuropeptide that mediates effects of suckling on maternal motivation, were abundant around galanin neurons in both preoptic regions. In the ACN and MPA, 89 and 82% of galanin neurons received close somatic appositions, with an average of 2.9 and 2.6 per cell, respectively. We observed perisomatic innervation of galanin neurons using correlated light and electron microscopy. The connection was excitatory based on the glutamate content of TIP39 terminals demonstrated by post-embedding immunogold electron microscopy. Injection of the anterograde tracer biotinylated dextran amine into the TIP39-expressing posterior intralaminar complex of the thalamus (PIL) demonstrated that preoptic TIP39 fibers originate in the PIL, which is activated by suckling. Thus, galanin neurons in the preoptic area of mother rats are innervated by an excitatory neuronal pathway that conveys suckling-related information. In turn, they can be topographically and neurochemically divided into two distinct cell groups, of which only one is affected by prolactin.

Ultrastructural Identification and Colocalization of Interacting Proteins in the Murine Cochlea by Post-Embedding Immunogold Transmission Electron Microscopy.

Verification of the presence and location of a protein within tissue can be accomplished by western blotting and immunohistochemistry, using either paraffin or frozen sections. Affinity purification by reciprocal coimmunoprecipitations using the tissue of interest can demonstrate the existence of an interacting pair of proteins. Ultimately, the ability to visualize the interaction at the cellular level is desired. Precise location(s) of interacting proteins in situ can be accomplished by ultrastructural localization with high-quality primary antibodies and small-particle-size Au-conjugated secondary antibodies. Visualization can be obtained with a transmission electron microscope fitted with a high-resolution camera permitting magnifications that exceed 2 × 10(5), and, to date, resolution capability of 20+ Mpixels, thus enabling localization of the target protein to within nanometers of the actual location. Here, we report the method by which immunolocalization at the level of the electron microscope is accomplished using the post-embedding technique, i.e., performing antibody labeling of proteins on ultrathin sections of tissue embedded in acrylic resin.

Region-specific changes in presynaptic agmatine and glutamate levels in the aged rat brain

During the normal aging process, the brain undergoes a range of biochemical and structural alterations, which may contribute to deterioration of sensory and cognitive functions. Age-related deficits are associated with altered efficacy of synaptic neurotransmission. Emerging evidence indicates that levels of agmatine, a putative neurotransmitter in the mammalian brain, are altered in a region-specific manner during the aging process. The gross tissue content of agmatine in the prefrontal cortex (PFC) of aged rat brains is decreased whereas levels in the temporal cortex (TE) are increased. However, it is not known whether these changes in gross tissue levels are also mirrored by changes in agmatine levels at synapses and thus could potentially contribute to altered synaptic function with age. In the present study, agmatine levels in presynaptic terminals in the PFC and TE regions (300 terminals/region) of young (3month; n=3) and aged (24month; n=3) brains of male Sprague–Dawley rats were compared using quantitative post-embedding immunogold electron-microscopy. Presynaptic levels of agmatine were significantly increased in the TE region (60%; p<0.001) of aged rats compared to young rats, however no significant differences were detected in synaptic levels in the PFC region. Double immunogold labeling indicated that agmatine and glutamate were co-localized in the same synaptic terminals, and quantitative analyses revealed significantly reduced glutamate levels in agmatine-immunopositive synaptic terminals in both regions in aged rats compared to young animals. This study, for the first time, demonstrates differential effects of aging on agmatine and glutamate in the presynaptic terminals of PFC and TE. Future research is required to understand the functional significance of these changes and the underlying mechanisms.

Immunoelectron microscope localization of androgen receptors and proliferating cell nuclear antigen in the epithelial cells of albino rat ventral prostate

Androgen receptor (AR) and proliferating cell nuclear antigen (PCNA) play a crucial role in development and progression of various prostatic diseases including prostatic carcinoma that is a leading cause of death in males. Previous studies have evaluated the expression pattern of AR and PCNA in prostate epithelial cells using immunohistochemistry (IHC). However, this technique has limited ability to identify their precise subcellular localization. Therefore, the aim of this study was to localize, subcellularly, AR and PCNA in the secretory epithelial cells of rat ventral prostate using post embedding immunogold-electron microscopy. The ventral lobes were dissected from six adult male albino rats after being perfused with paraformaldehyde. Some specimens were immuno-labeled with AR or PCNA and others were processed for immunoelectron microscope of AR and PCNA using 15-nm gold conjugated secondary antibodies. The results showed that, by immunoperoxidase reaction, AR and PCNA were localized diffusely throughout the nuclei of the epithelial cells of prostatic acini without visible cytoplasmic expression. However, the higher resolution immuno-electron microscopy was able to detect AR and PCNA in the nucleus and some cytoplasmic organelles. In conclusion, this study emphasizes the importance of immuoelectron microscopy in precise localization of AR and PCNA at the subcelullar levels in the secretory epithelial cells of the rat prostatic acini. These findings will help to further understand the mechanism of action of these receptors under normal and pathological conditions that could have future clinical application after careful human investigation.

The organization of AMPA receptor subunits at the postsynaptic membrane

AMPA receptors are the principal mediators of excitatory synaptic transmission in the mammalian central nervous system. The subunit composition of these tetrameric receptors helps to define their functional properties, and may also influence the synaptic trafficking implicated in long-term synaptic plasticity. However, the organization of AMPAR subunits within the synapse remains unclear. Here, we use postembedding immunogold electron microscopy to study the synaptic organization of AMPAR subunits in stratum radiatum of CA1 hippocampus in the adult rat. We find that GluA1 concentrates away from the center of the synapse, extending at least 25 nm beyond the synaptic specialization; in contrast, GluA3 is uniformly distributed along the synapse, and seldom extends beyond its lateral border. The fraction of extrasynaptic GluA1 is markedly higher in small than in large synapses; no such effect is seen for GluA3. These observations imply that different kinds of AMPARs are differently trafficked to and/or anchored at the synapse.

Alterations of monocarboxylate transporter densities during hypoxia in brain and breast tumour cells

BackgroundTumour cells are characterized by aerobic glycolysis, which provides biomass for tumour proliferation and leads to extracellular acidification through efflux of lactate via monocarboxylate transporters (MCTs). Deficient and spasm-prone tumour vasculature causes variable hypoxia, which favours tumour cell survival and metastases. Brain metastases frequently occur in patients with advanced breast cancer.Effective treatment strategies are therefore needed against brain metastasis from breast carcinoma.Material and methodsIn order to identify differences in the capacity for lactate exchange, human T-47D breast cancer cells and human glioblastoma T98G cells were grown under 4 % or 20 % oxygen conditions and examined for MCT1, MCT2 and MCT4 expression on plasma membranes by quantitative post embedding immunogold electron microscopy. Whereas previous studies on MCT expression in tumours have recorded mRNA and protein levels in cell extracts, we examined concentrations of the proteins in the microvillous plasma membrane protrusions specialized for transmembrane transport.ResultsIn normoxia, both tumour cell types highly expressed the low affinity transporter MCT4, which is thought to mainly mediate monocarboxylate efflux, while for high affinity transport the breast tumour cells preferentially expressed MCT1 and the brain tumour cells resembled brain neurons in expressing MCT2, rather than MCT1. The expressions of MCT1 and MCT4 were upregulated in hypoxic conditions in both breast and brain tumour cells. The expression of MCT2 also increased in hypoxic breast cancer cells, but decreased in hypoxic brain tumour cells. Quantitative immunoblots showed similar hypoxia induced changes in the protein levels.ConclusionThe differential expression and regulation of MCTs in the surface membranes of hypoxic and normoxic tumour cells of different types provide a foundation for innovation in tumour therapy through the selective targeting of MCTs. Selective inhibition of various MCTs could be an efficient way to quench an important energy source in both original breast tumour and metastatic cancer tissue in the brain.

Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II Signaling within Hippocampal Glutamatergic Postsynapses during Flurazepam Withdrawal

Cessation of one-week oral administration of the benzodiazepine flurazepam (FZP) to rats results in withdrawal anxiety after 1 day of withdrawal. FZP withdrawal is correlated with synaptic incorporation of homomeric GluA1-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) in the proximal stratum radiatum of CA1 neurons. After 2 days of withdrawal, Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates GluA1 subunits at Ser831, increasing channel conductance. Secondary to AMPAR potentiation, GluN2B-containing N-methyl-D-aspartate receptors (NMDARs), known binding partners of CaMKII, are selectively removed from the postsynaptic density (PSD). While activation of synaptic CaMKII is known to involve translocation to the PSD, CaMKII bound to NMDARs may be removed from the PSD. To distinguish these possibilities, the current studies used postembedding immunogold electron microscopy to investigate alterations in CaMKII signaling at CA1 stratum radiatum synapses after 2 days of FZP withdrawal. These studies revealed decreased total, but not autophosphorylated (Thr286) CaMKIIα expression in CA1 PSDs. The removal of CaMKII-GluN2B complexes from the PSD during drug withdrawal may serve as a homeostatic mechanism to limit AMPAR-mediated CA1 neuron hyperexcitability and benzodiazepine withdrawal anxiety.

Glutamate Receptor δ2 Is Essential for Input Pathway-Dependent Regulation of Synaptic AMPAR Contents in Cerebellar Purkinje Cells

The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor δ2 (GluRδ2 or GluD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRδ2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluRδ2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluRδ2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluRδ2. These results suggest that GluRδ2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.

MHC class I molecules are present both pre- and postsynaptically in the visual cortex during postnatal development and in adulthood

Immune molecules have been discovered recently to play critical roles in the development, function, and plasticity of the cerebral cortex. MHC class I (MHCI) molecules are expressed in the central nervous system and regulate activity-dependent refinement of visual projections during late postnatal development. They have also been implicated in neurodevelopmental diseases such as schizophrenia and autism. Despite the excitement generated by these unique roles for immune proteins in the brain, little is known about how these molecules regulate cortical connections. The first step toward elucidating the mechanism is to identify the spatial and temporal distribution of MHCI proteins throughout development. Using a pan-specific antibody that recognizes many MHCI variants for biochemistry and immunohistochemistry, we found that MHCI proteins are expressed in the rat visual cortex at all ages examined-during the peak of synaptogenesis, the critical period of synaptic refinement, and adulthood. Their abundance in the cortex peaked during early postnatal development, declining during periods of plasticity and adulthood. In contrast to current assumptions, pre- and postembedding immunogold electron microscopy (EM) revealed that MHCI proteins were present both pre- and postsynaptically at all ages examined. They were often found in the postsynaptic density and were closely associated with synaptic vesicles in the presynaptic terminal. These results suggest a previously undescribed model in which MHCI molecules function on both sides of the synapse to regulate connectivity in the mammalian visual cortex before, during, and after the establishment of connections.

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.

Organization of NMDA receptors at extrasynaptic locations

NMDA receptors are found in neurons both at synapses and in extrasynaptic locations. Extrasynaptic locations are poorly characterized. Here we used preembedding immunoperoxidase and postembedding immunogold electron microscopy and fluorescence light microscopy to characterize extrasynaptic NMDA receptor locations in dissociated hippocampal neurons in vitro and in the adult and postnatal hippocampus in vivo. We found that extrasynaptic NMDA receptors on neurons in vivo and in vitro were usually concentrated at points of contact with adjacent processes, which were mainly axons, axon terminals, or glia. Many of these contacts were shown to contain adhesion factors such as cadherin and catenin. We also found associations of extrasynaptic NMDA receptors with the membrane associated guanylate kinase (MAGUKs), postsynaptic density (PSD)-95 and SAP102. Developmental differences were also observed. At postnatal day 2 in vivo, extrasynaptic NMDA receptors could often be found at sites with distinct densities whereas dense material was seen only rarely at sites of extrasynaptic NMDA receptors in the adult hippocampus in vivo. This difference probably indicates that many sites of extrasynaptic NMDA receptors in early postnatal ages represent synapse formation or possibly sites for synapse elimination. At all ages, as suggested in both in vivo and in vitro studies, extrasynaptic NMDA receptors on dendrites or the sides of spines may form complexes with other proteins, in many cases, at stable associations with adjacent cell processes. These associations may facilitate unique functions for extrasynaptic NMDA receptors.

Positive allosteric activation of GABAA receptors bi-directionally modulates hippocampal glutamate plasticity and behaviour

Long-term BZ (benzodiazepine) anxiolytic therapy increases the risk of physical dependence manifested as withdrawal anxiety. BZ-induced potentiation of GABA(A)R (gamma-aminobutyric acid type-A receptor) function by 1-week oral administration of FZP (flurazepam) bi-directionally modulates excitatory glutamatergic synaptic transmission in hippocampal CA1 neurons during drug withdrawal. Previous electrophysiological studies on acutely isolated and intact CA1 neurons, as well as immunofluorescence and post-embedding immunogold electron microscopy studies, suggest increased synaptic insertion of GluR (glutamate receptor) 2-lacking AMPARs (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors) in 2-day FZP-withdrawn rats. Preliminary studies indicated a similar increase in GluR1, then phospho-Ser(831)-GluR1, as well as CaMKIIalpha (Ca(2+)/calmodulin-dependent protein kinase IIalpha), but not phospho-Thr(286)-CaMKII levels at the same time point. In our studies, whole-cell recordings in hippocampal slices revealed that AMPAR mEPSC [miniature EPSC (excitatory postsynaptic current)] amplitude was increased in 1-day FZP-withdrawn rats followed by an increase in estimated single-channel conductance in 2-day-FZP-withdrawn rats. Enhanced conductance was no longer observed in slices pre-incubated for 2 h in the CaMKII inhibitor KN-93, but not the inactive analogue KN-92. To evaluate whether CaMKII-mediated AMPA potentiation could occlude LTP (long-term potentiation), LTP was induced by TBS (theta burst stimulation) and recorded using whole-cell and extracellular techniques. LTP was induced in both groups, but only maintained for <15 min in 2-day FZP-withdrawn rats. LTP was fully restored after 7-day withdrawal. Despite the lack of LTP maintenance, impairment of object recognition, place and context was not observed in 2-day-FZP-withdrawn rats. Since L-VGCC (L-type voltage-gated calcium channel) current density was doubled on drug withdrawal and up to 2 days, Ca(2+) entry through L-VGCCs and perhaps subsequently through Ca(2+)-permeable AMPARs are proposed to be responsible for enhanced CaMKIIalpha levels and AMPAR potentiation. Mechanisms associated with several different models of activity-dependent plasticity may underlie BZ physical dependence.

Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses

The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.