Electron tomography on γ-aminobutyric acid-ergic synapses reveals a discontinuous postsynaptic network of filaments.

The regulation of synaptic strength at γ-aminobutyric acid (GABA)-ergic synapses is dependent on the dynamic capture, retention, and modulation of GABA A-type receptors by cytoplasmic proteins at GABAergic postsynaptic sites. How these proteins are oriented and organized in the postsynaptic cytoplasm is not yet established. To better understand these structures and gain further insight into the mechanisms by which they regulate receptor populations at postsynaptic sites, we utilized electron tomography to examine GABAergic synapses in dissociated rat hippocampal cultures. GABAergic synapses were identified and selected for tomography by using a set of criteria derived from the structure of immunogold-labeled GABAergic synapses. Tomography revealed a complex postsynaptic network composed of filaments that extend ∼ 100 nm into the cytoplasm from the postsynaptic membrane. The distribution of these postsynaptic filaments was strikingly similar to that of the immunogold label for gephyrin. Filaments were interconnected through uniform patterns of contact, forming complexes composed of 2-12 filaments each. Complexes did not link to form an integrated, continuous scaffold, suggesting that GABAergic postsynaptic specializations are less rigidly organized than glutamatergic postsynaptic densities.

Glutamatergic lateral parabrachial neurons innervate orexin-containing hypothalamic neurons in the rat.

We performed this study to understand the anatomical substrates of parabrachial nucleus (PBN) modulation of orexin (ORX)-containing neurons in the hypothalamus. After biotinylated dextranamine (BDA) injection into the lateral PBN and immunostaining of ORX-containing neurons in the rat, the prominent overlap of the distribution field of the BDA-labeled fibers and that of the ORX-immunoreactive (ir) neurons was found in the lateralmost part of the dorsomedial nucleus and adjacent dorsal perifornical area (this overlapping field was referred to as "suprafornical area" in the present study), and the labeled axon terminals made asymmetrical synaptic contacts with somata and dendrites of the ORX-ir neurons. We further revealed that almost all the "suprafornical area"-projecting lateral PBN neurons were positive for vesicular glutamate transporter 2 mRNA and very few of them were positive for glutamic acid decarboxylase 67 mRNA. The present data suggest that ORX-containing neurons in the "suprafornical area" may be under the excitatory influence of the glutamatergic lateral PBN neurons probably for the regulation of arousal and waking.

Glutamate decarboxylase-derived IDDM autoantigens displayed on self-assembled protein nanoparticles.

The recombinant ferritin heavy chain (FTN-H) formed self-assembled spherical nanoparticles with the size comparable to native one. We tried to express the GAD65 COOH-terminal fragments, i.e., 448-585 (GAD65(448-585)), 487-585 (GAD65(487-585)), and 512-585 (GAD65(512-585)) amino acid fragments, using FTN-H as N-terminus fusion expression partner in Escherichia coli. All of recombinant fusion proteins (FTN-H::GAD65(448-585), FTN-H::GAD65(487-585), and FTN-H::GAD65(512-585)) also formed spherical nanoparticles due probably to the self-assembly function of the fused ferritin heavy chain. The antigenic epitopes within GAD65(448-585), GAD65(487-585), and GAD65(512-585) against insulin-dependent diabetes mellitus (IDDM) marker (autoantibodies against GAD65) were localized at the surface of the spherical protein nanoparticles so that anti-GAD65 Ab could recognize them. Protein nanoparticles like FTN-H seem to provide distinct advantages over other inorganic nanoparticles (e.g., Au, Ag, CdSe, etc.) in that through the bacterial synthesis, the active capture probes can be located at the nanoparticle surface with constant orientation/conformation via covalent cross-linking without complex chemistry. Also it is possible for the protein nanoparticles to have uniform particle size, which is rarely achieved in the chemical synthesis of inorganic nanoparticles. Thus, the recombinant ferritin particles can be used as a three-dimensional (spherical) and nanometer-scale probe structure that is a key component in ultra-sensitive protein chip for detecting protein-small molecule interactions and protein-protein interactions.

Synaptic vesicle protein synaptoporin is differently expressed by subpopulations of mouse hippocampal neurons.

In the hippocampus, the synaptic vesicle protein synaptoporin (SPO) has been reported to be exclusively enriched in the granule cell axons, the mossy fibers. In this study, we show that in adult rats and mice SPO immunoreactivity (IR) is also detectable in strata oriens, radiatum, and lacunosum-moleculare of CA1-CA3, as well as perisomatically in the hippocampus proper and fascia dentata. In situ hybridization confirmed that SPO mRNA was present in granule cells and CA3 pyramidal cells but not in CA1 pyramidal cells. Importantly, cells scattered throughout the hippocampal layers resembling the distribution of interneurons were found to synthesize high amounts of SPO mRNA, too. Thus, these findings indicate that SPO expression in the hippocampus was underestimated until now. Moreover, double-labeling immunohistochemistry and confocal microscopy revealed selective colocalization of SPO and glutamate decarboxylase (GAD 65), a marker for gamma-aminobutyric acid (GABA)ergic terminals. To identify SPO expressing interneurons, in situ hybridization was combined with immunocytochemistry against parvalbumin (PV), calbindin (CB), calretinin (CR), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP). We found that SPO transcripts were differentially expressed by various interneuron subpopulations in the hippocampus of C57Bl/6 mice (PV 44.2%, CB 46.3%, CR 19.3%, CCK 38.6%, VIP 59.9%). Immunoelectron microscopy for SPO labeled synaptic vesicle profiles in distinct symmetric and asymmetric synapses. In conclusion, our data demonstrate that hippocampal principal cells and interneurons display a variety of synaptic vesicles that are likely to contribute to the functional characteristics of their output synapses.