Antibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopy.

BACKGROUND: Antibodies are one of the most important tools in biological research. High specificity and sensitivity of antibodies are crucial to obtain reliable results. Tissue fixed with glutaraldehyde (GA) is commonly used in electron microscopical investigations. The fixation and embedding routine in preparation of tissue for post-embedding electron microscopy (EM) will mask and structurally alter epitopes, making antibody-antigen interaction inefficient, with low labeling intensities. One of the main factors in this regard is the use of GA as fixative. NEW METHOD: To alleviate these technical challenges, we immunized rabbits with antigen pre-fixed with GA. We hypothesized that the resulting antibodies would have stronger affinity to antigens that have been conformationally changed and denatured by GA, the way they are in fixed tissue. COMPARISON WITH EXISTING METHOD AND RESULTS: An initial screening with western blotting (WB) showed results consistent with our hypothesis. In-house antibodies raised against GA-fixed SNARE proteins SNAP-25 and VAMP2, binds more strongly to fixed proteins compared to non-fixed proteins, while the pattern is opposite with the commercially available antibodies raised against non-fixed antigens (standard antibodies). Quantitative post-embedding EM of hippocampal synapses gave higher labeling intensities with anti-GA-SNAP-25 and anti-GA-VAMP2 compared to standard antibodies. Importantly, light microscopy (LM) and EM with our antibodies revealed stronger labeling of GA-fixed than formaldehyde (FH) treated brains. CONCLUSION: Our results highlight the experimental potential of raising antibodies against GA-treated antigen to improve sensitivity of the antibodies for postembedding immunogold EM.

Correction to: A possible postsynaptic role for SNAP-25 in hippocampal synapses.

In the original publication of the article the author name M. Schupps was incorrect.

A possible postsynaptic role for SNAP-25 in hippocampal synapses.

The SNARE protein SNAP-25 is well documented as regulator of presynaptic vesicle exocytosis. Increasing evidence suggests roles for SNARE proteins in postsynaptic trafficking of glutamate receptors as a basic mechanism in synaptic plasticity. Despite these indications, detailed quantitative subsynaptic localization studies of SNAP-25 have never been performed. Here, we provide novel electron microscopic data of SNAP-25 localization in postsynaptic spines. In addition to its expected presynaptic localization, we show that the protein is also present in the postsynaptic density (PSD), the postsynaptic lateral membrane and on small vesicles in the postsynaptic cytoplasm. We further investigated possible changes in synaptic SNAP-25 protein expression after hippocampal long-term potentiation (LTP). Quantitative analysis of immunogold-labeled electron microscopy sections did not show statistically significant changes of SNAP-25 gold particle densities 1 h after LTP induction, indicating that local trafficking of SNAP-25 does not play a role in the early phases of LTP. However, the strong expression of SNAP-25 in postsynaptic plasma membranes suggests a function of the protein in postsynaptic vesicle exocytosis and a possible role in hippocampal synaptic plasticity.

Presynaptic PICK1 facilitates trafficking of AMPA-receptors between active zone and synaptic vesicle pool.

Previous studies have indicated that presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) contribute to the regulation of neurotransmitter release. In hippocampal synapses, the presynaptic surface expression of several AMPAR subunits, including GluA2, is regulated in a ligand-dependent manner. However, the molecular mechanisms underlying the presynaptic trafficking of AMPARs are still unknown. Here, using bright-field immunocytochemistry, western blots, and quantitative immunogold electron microscopy of the hippocampal CA1 area from intact adult rat brain, we demonstrate the association of AMPA receptors with the presynaptic active zone and with small presynaptic vesicles, in Schaffer collateral synapses in CA1 of the hippocampus. Furthermore, we show that GluA2 and protein interacting with C kinase 1 (PICK1) are colocalized at presynaptic vesicles. Similar to postsynaptic mechanisms, overexpression of either PICK1 or pep2m, which inhibit the N-ethylmaleimide sensitive fusion protein (NSF)-GluA2 interaction, decreases the concentration of GluA2 in the presynaptic active zone membrane. These data suggest that the interacting proteins PICK1 and NSF act as regulators of presynaptic GluA2-containing AMPAR trafficking between the active zone and a vesicle pool that may provide the basis of presynaptic components of synaptic plasticity.

The discovery of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and the molecular regulation of synaptic vesicle transmitter release: the 2010 Kavli Prize in neuroscience.

Brain function depends on a crucial feature: The ability of individual neurons to share packets of information, known as quantal transmission. Given the sheer number of tasks the brain has to deal with, this information sharing must be extremely rapid. Synapses are specialized points of contact between neurons, where fast transmission takes place. Though the basic elements and functions of the synapse had been established since the 1950s, the molecular basis for regulation of fast synaptic transmitter release was not known 20 years ago. However, around 1990, crucial discoveries were made by Richard Scheller, James Rothman, and Thomas Südhof, leading a few years later to the formulation of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) hypothesis and a new understanding of the molecular events controlling vesicular release of transmitter in synapses. The 2010 Kavli Prize in neuroscience was awarded to these three researchers, "for their work to reveal the precise molecular basis of the transfer of signals between nerve cells in the brain."

Protein interacting with C kinase 1 (PICK1) and GluR2 are associated with presynaptic plasma membrane and vesicles in hippocampal excitatory synapses.

AMPA receptors have been identified in different populations of presynaptic terminals and found to be involved in the modulation of neurotransmitter release. The mechanisms that govern the expression of presynaptic AMPA receptors are not known. One possibility is that pre- and postsynaptic AMPA receptors are regulated according to the same principles. To address this hypothesis we investigated whether protein interacting with C kinase 1 (PICK1), known to interact with AMPA receptors postsynaptically, also is expressed presynaptically, together with AMPA receptors. Subfractionation and high-resolution immunogold analyses of the rat hippocampus revealed that GluR2 and PICK1 are enriched postsynaptically, but also in presynaptic membrane compartments, including the active zone and vesicular membranes. PICK1 and GluR2 are associated with the same vesicles, which are immunopositive also for synaptophysin and vesicle-associated membrane protein 2. Based on what is known about the function of PICK1 postsynaptically, the present data suggest that PICK1 is involved in the regulation of presynaptic AMPA receptor trafficking and in determining the size of the AMPA receptor pool that modulates presynaptic glutamate release.

Neuronal enriched endosomal protein of 21 kDa colocalizes with glutamate receptor subunit GLUR2/3 at the postsynaptic membrane.

Functional evidence suggests that neuronal enriched endosomal protein of 21 kDa (NEEP21) takes part in facilitating transport of AMPA receptors (AMPAR) in the synapse. To explore the anatomical basis for a role in this synaptic trafficking, we investigated the ultrastructural localization of NEEP21 in rodent brain. Using immunogold electron microscopy, we show that NEEP21 is colocalized with the AMPAR subunits GluR2/3 in postsynaptic spines. Quantitative analysis of gold particle distribution along an axis perpendicular to the postsynaptic specialization indicated that NEEP21 occurs in the postsynaptic membrane but also in the interior of the spines. NEEP21 positive endosomes/multivesicular bodies were found throughout cell bodies and dendrites. In light microscopical preparations, the NEEP21 antibody produced a labeling pattern in the neocortex, hippocampus and cerebellum that mimicked that of GluR2/3 and not that of GluR1 or 4. Our findings are consistent with a role for NEEP21 in facilitating vesicular transport of GluR2 between intracellular compartments and the postsynaptic plasma membrane.

Antibodies to CRMP3-4 associated with limbic encephalitis and thymoma.

We present a case with subacute limbic encephalitis (LE) and thymoma. Neither classical onconeural antibodies nor antibodies to voltage gated potassium channels (VGKC) were detected, but the serum was positive for anti-glutamic acid decarboxylase (GAD). The patient serum also stained synaptic boutons of pyramidal cells and nuclei of granule cells of rat hippocampus. The objective of the study was to identify new antibodies associated with LE. Screening a cDNA expression library identified collapsin response mediator protein 3 (CRMP3), a protein involved in neurite outgrowth. The serum also reacted with both CRMP3 and CRMP4 by Western blot. Similar binding pattern of hippocampal granule cells was obtained with the patient serum and rabbit anti-serum against CRMP1-4. The CRMP1-4 antibodies stained neuronal nuclei of a biopsy from the patient's temporal lobe, but CRMP1-4 expression in thymoma could only be detected by immunoblotting. Absorption studies with recombinant GAD failed to abolish the staining of the hippocampal granule cells. Our findings illustrate that CRMP3-4 antibodies can be associated with LE and thymoma. This has previously been associated with CRMP5.

Sec6 is localized to the plasma membrane of mature synaptic terminals and is transported with secretogranin II-containing vesicles.

The sec6/8 (exocyst) complex is implicated in targeting of vesicles for regulated exocytosis in various cell types and is believed to play a role in synaptogenesis and brain development. We show that the subunits sec6 and sec8 are present at significant levels in neurons of adult rat brain, and that immunoreactivity for the two subunits has a differential subcellular distribution. We show that in developing as well as mature neurons sec6 is concentrated at the inside of the presynaptic plasma membrane, while sec8 immunoreactivity shows a diffuse cytoplasmic distribution. Among established, strongly synaptophysin-positive neuronal boutons, sec6 displays highly differential concentrations, indicating a role for the complex independent of the ongoing synaptic-vesicle release activity. Sec6 is transported along neurites on secretogranin II-positive vesicles, while sec6-negative/secretogranin II-positive vesicles stay in the cell body. In PC12 cells, sec6-positive vesicles accumulate at the plasma membrane at sites of cell-cell contact. Neuronal induction of the PC12 cells with nerve growth factor shows that sec8 is not freely soluble, but may probably interact with cytoskeletal elements. The complex may facilitate the targeting of membrane material to presynaptic sites and may possibly shuttle vesicles from the cytoskeletal transport machinery to presynaptic membrane sites. Thus, we suggest that the exocyst complex serves to modulate exocytotic activity, by targeting membrane material to its presynaptic destination.

Hrs-2 regulates receptor-mediated endocytosis via interactions with Eps15.

Hrs-2, via interactions with SNAP-25, plays a regulatory role on the exocytic machinery. We now show that Hrs-2 physically interacts with Eps15, a protein required for receptor-mediated endocytosis. The Hrs-2/Eps15 interaction is calcium dependent, inhibited by SNAP-25 and alpha-adaptin, and results in the inhibition of receptor-mediated endocytosis. Immunoelectron microscopy reveals Hrs-2 localization on the limiting membrane of multivesicular bodies, organelles in the endosomal pathway. These data show that Hrs-2 regulates endocytosis, delineate a biochemical pathway (Hrs-2-Eps15-AP2) in which Hrs-2 functions, and suggest that Hrs-2 acts to provide communication between endo- and exocytic processes.

The cellular and developmental expression of hrs-2 in rat.

The molecular events underlying vesicular trafficking probably involve the formation and dissolution of protein complexes between integral components of the vesicle and its target membrane. SNAP-25 is associated with the plasma membrane and is a component of a core protein complex thought to be essential for neurotransmitter release. We have previously characterized a protein, hrs-2, that interacts with SNAP-25 and inhibits secretion from permeabilized PC12 cells. The cellular localization and developmental expression patterns of a number of proteins involved in the secretion machinery have been documented. To understand more about the possible cellular role of hrs-2, we have examined hrs-2 distribution, developmental expression and subcellular localization in rat tissues and cell lines. We show herein that the distribution of hrs-2 in brain and periphery parallels that of SNAP-23/25, and that recombinant hrs-2 binds to both SNAP-23 and SNAP-25. Hrs-2 mRNA and protein are found almost ubiquitously in neurons in the brain. Hrs-2 mRNA is expressed in the neural tube at E10 and thereafter mRNA and protein levels remain relatively constant in the whole brain through adulthood. In cultured PC12 cells, endogenous hrs-2 is expressed in the cytoplasm and on the limiting membranes of multivesicular bodies. Overexpression of hrs-2 in mammalian cells results in the appearance of large intracellular compartments that are labelled with hrs-2 antibodies. The wide distribution, the interaction with SNAP-23 and the localization on multivesicular body membranes suggest a general role for hrs-2 in cellular machinery.

Syntaxin 6 functions in trans-Golgi network vesicle trafficking.

The specific transfer of vesicles between organelles is critical in generating and maintaining the organization of membrane compartments within cells. Syntaxin 6 is a recently discovered member of the syntaxin family, whose constituents are required components of several vesicle trafficking pathways. To better understand the function of syntaxin 6, we generated a panel of monoclonal antibodies that specifically recognize different epitopes of the protein. Immunoelectron microscopy shows syntaxin 6 primarily on the trans-Golgi network (TGN), where is partially colocalizes with the TGN adapter protein AP-1 on clathrin-coated membranes. Additional label is present on small vesicles in the vicinity of endosome-like structures. Immunoprecipitation of syntaxin 6 revealed that it is present in a complex or complexes with alpha-soluble NSF attachment protein, vesicle-associated membrane protein 2, or cellubrevin and a mammalian homologue of VPS45, which is a member of the sec1 family implicated in Golgi to prevacuolar compartment trafficking in yeast. We show that mammalian VPS45 is found in multiple tissues, is partially membrane associated, and is enriched in the Golgi region. Converging lines of evidence suggest that syntaxin 6 mediates a TGN trafficking event, perhaps targeting to endosomes in mammalian cells.

Localization of the glutamate transporter protein GLAST in rat retina.

Glutamate is a neurotransmitter in retina. Glutamate transporter proteins keep the resting extracellular glutamate concentration low. This is required for normal neurotransmission and prevents the extracellular concentration of glutamate from reaching toxic levels. Here we describe the light and electron microscopic localization of the glutamate transporter protein GLAST in rat retina using an antibody raised and affinity purified against a peptide corresponding to amino acid residues 522-541. The strongest immunocytochemical labelling was observed in the outer plexiform layer, ganglion cell layer, and optic disc. GLAST was found in Müller cell processes in all retinal layers, notably ensheathing the photoreceptor terminals in the outer plexiform layer, and in astrocytes close to vessels in the inner retina and optic disc. No labelling was observed in neurons. The electrophoretic mobility of GLAST in retina was similar to that in cerebellum. In conclusion, the findings are in agreement with those reported by Derouiche and Rauen [7], except that we did not detect any GLAST in the retinal pigment epithelium.

The mammalian brain rsec6/8 complex.

rsec6 and rsec8 are two components of a 17S complex in mammalian brain that is homologous to the yeast 834 kDa Sec6/8/15 complex which is essential for exocytosis. Purification and partial amino acid sequencing of the mammalian rsec6/8 complex reveals that it is composed of eight novel proteins with a combined molecular weight of 743 kDa. The complex is broadly expressed in brain and displays a plasma membrane localization in nerve terminals. Membrane associated rsec6/8 complex coimmunoprecipitates with syntaxin, a plasma membrane protein critical for neurotransmission. These data suggest a role for the mammalian rsec6/8 complex in neurotransmitter release via interactions with the core vesicle docking and fusion apparatus.

Colocalization of amino acid signal molecules in neurons and endocrine cells.

During the last 20 to 30 years, numerous examples have been provided of neurons and endocrine cells that are able to produce, store, and in many cases release more than one type of signal molecule. Recent models propose that neurons often employ an amino acid, an amine, and one or more neuroactive peptides, and that endocrine cells may release more than one peptide hormone. In neurons, the different classes of transmitter convey fast, intermediate, and slow signalling respectively. However, a series of studies demonstrates that neurons may colocalize more than one neuroactive amino acid, and that endocrine cells may contain an amino acid along with their peptide hormone. These forms of colocalization seem to add new levels of complexity to the role of amino acids in cell signalling, suggesting that, in neurons, amino acids may interact at the receptor level, modifying the effect of each other, and that, in endocrine cells, amino acids may act together with or parallel to a peptide hormone in a paracrine or autocrine manner.

Organization of AMPA receptor subunits at a glutamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.

Sensitive and high-resolution immunocytochemical procedures were used to investigate the spatial organization of AMPA receptor subunits (GluR1-4) at the synapse between the inner hair cells and the afferent dendrites in the rat organ of Corti. This is a synapse with special functional properties and with a presynaptic dense body that defines the center of the synapse and facilitates its morphometric analysis. A quantitative postembedding immunocytochemical analysis was performed on specimens that had been embedded in a metachrylate resin at low temperature after freeze substitution. Single- and double-labeling procedures indicated that GluR2/3 and GluR4 subunits were colocalized throughout the postsynaptic density, with a maximum distance of 300 nm from the presynaptic body and with higher concentrations peripherally than centrally. No receptor immunolabeling was found at extrasynaptic membranes, but some GluR4 subunits appeared to be expressed presynaptically. The synapses between outer hair cells and afferent dendrites were devoid of labeling. The present data indicate that AMPA receptor subunits are inserted into the postsynaptic membrane in a very precise manner and that their density increases on moving away from the center of the synapse.

Co-localization of glutamate and homocysteic acid immunoreactivities in human photoreceptor terminals.

Consecutive semithin sections of human retinae were treated with antisera recognizing fixed homocysteic acid, glutamate or glutamine. Photoreceptor terminals displayed a co-localization of glutamate-like and homocysteic acid-like immunoreactivities. This was confirmed in the electron microscope by immunogold cytochemistry. A quantitative analysis of the immunogold labelling indicated that glutamate and homocysteic acid occurred at higher concentrations in the terminals than in outer parts of the receptor cells. No such gradient was found for glutamine immunoreactivity, which was concentrated in Müller cell processes. These processes were also labelled by the homocysteic acid antiserum, although less intensely than were the photoreceptor terminals. Control experiments suggested that the homocysteic acid antiserum visualized a pool of authentic homocysteic acid, although it could not be excluded that part of this pool had been generated by non-enzymatic oxidation of precursor molecules. Homocysteic acid immunoreactivity was also demonstrated in photoreceptor terminals of baboon. The present data indicate that primate photoreceptor terminals contain homocysteic acid in addition to glutamate and open up the possibility that homocysteic acid is released as a glutamate co-agonist at photoreceptor synapses.

Colocalization of gamma-aminobutyrate and gastrin in the rat antrum: an immunocytochemical and in situ hybridization study.

BACKGROUND/AIMS: The inhibitory neurotransmitter gamma-aminobutyrate (GABA) has been shown to coexist with insulin in pancreatic beta-cells. We have presently investigated whether GABA also colocalizes with gastrin in G cells in rat antral mucosa. METHODS: Three alternative approaches were used: (1) gastrin in situ hybridization and GABA immunocytochemistry on consecutive cryostat sections; (2) GABA immunocytochemistry and gastrin immunocytochemistry on adjacent semithin and ultrathin sections; and (3) double-immunogold labeling of GABA and gastrin in the same ultrathin section. RESULTS: Colocalization of GABA and gastrin was observed with each of the three approaches. In the double-immunogold labeled cells, the G-cell granules displayed a high gold-particle density indicating gastrin and a low particle density indicating GABA, whereas the converse was true for the extragranular cytoplasmic matrix. The gold-particle ratios between these compartments were 11 (for gastrin) and 0.36 (for GABA), respectively. GABA labeling was also observed in two other antral endocrine cell types, classified by morphological criteria as somatostatin producing D cells and serotonin producing ECn cells. CONCLUSIONS: This is the first direct demonstration of GABA in gastrointestinal G cells. Our findings suggest that GABA may have a paracrine function in the stomach mucosa, analogous to its presumed role in the pancreatic islets.

Distribution of GABA immunoreactivity in kainic acid-treated rabbit retina.

Ischaemic retinal cell degeneration seems to involve both NMDA and non-NMDA receptor over-stimulation. However, different retinal cell types differ largely in their susceptibility to excitatory amino acid-induced neurotoxicity. We have investigated the vulnerability of GABAergic cells in the rabbit retina to the non-NMDA receptor agonist kainic acid (KA). The distribution of GABA immunoreactivity (GABA-IR) was examined in the central inferior retina at different survival times (5 h-6 days) following an intra-ocular injection of 140 nmol KA and compared to that of control and untreated retinas. In the normal retina, the majority of GABA-positive cells (79%) were located in the inner nuclear layer (INL), in one to four cell rows next to the inner plexiform layer (IPL), and in one cell row next to the outer plexiform layer (OPL). The remainder (21%) were found in the ganglion cell layer (GCL). Dense immunoreactivity was seen throughout the IPL. In the OPL, stained dots and occasional immunoreactive large processes could be seen. KA-exposed retinas processed for GABA immunocytochemistry 5 and 24 h after the injection showed an 85% reduction in the number of GABA immunoreactive cells. About the same degree of depletion was seen among GABA-IR cells located at different retinal levels. However, at these survival times, immunostaining was observed in three distinct bands in the IPL, indicating that the vulnerability to KA is not uniformly distributed among all GABAergic cells. At 48 h, an additional decrease in the number of labelled cells was noted, but immunoreactive cells were still found both in the INL and GCL. Even 6 days after KA treatment, a few stained cell bodies were seen in the INL next to the IPL, as well as a few processes in the IPL. The study shows that KA receptor overstimulation induces a marked depletion of the endogenous cellular GABA pools of the central rabbit retina, most likely as a result of GABAergic cell loss. However, a small population of GABAergic cells located in the INL appears to be less vulnerable to the toxic effects of 140 nmol KA.