Structural organization of the presynaptic density at identified synapses in the locust central nervous system

Abstract

In a synaptic active zone, vesicles aggregate around a densely staining structure called the presynaptic density. We focus on its three-dimensional architecture and a major molecular component in the locust. We used electron tomography to study the presynaptic density in synapses made in the brain by identified second-order neuron of the ocelli. Here, vesicles close to the active zone are organized in two rows on either side of the presynaptic density, a level of organization not previously reported in insect central synapses. The row of vesicles that is closest to the density's base includes vesicles docked with the presynaptic membrane and thus presumably ready for release, whereas the outer row of vesicles does not include any that are docked. We show that a locust ortholog of the Drosophila protein Bruchpilot is localized to the presynaptic density, both in the ocellar pathway and compound eye visual neurons. An antibody recognizing the C-terminus of the Bruchpilot ortholog selectively labels filamentous extensions of the presynaptic density that reach out toward vesicles. Previous studies on Bruchpilot have focused on its role in neuromuscular junctions in Drosophila, and our study shows it is also a major functional component of presynaptic densities in the central nervous system of an evolutionarily distant insect. Our study thus reveals Bruchpilot executes similar functions in synapses that can sustain transmission of small graded potentials as well as those relaying large, spike-evoked signals. J. Comp. Neurol. 520:384–400, 2012. © 2011 Wiley Periodicals, Inc.

Synaptic connections of first‐stage visual neurons in the locust Schistocerca gregaria extend evolution of tetrad synapses back 200 million years

“…Synapses on block face scanning electron micrographs are identifiable because an electron‐dense presynaptic bar reaches into the presynaptic cell (Leitinger et al, ); the bar and its surrounding vesicles are visible as a darkening in the cytoplasm of the presynaptic cell. Identifying synapses with intracellular cues from SBEM micrographs is novel for the invertebrate nervous system, so the question was whether the quality of the SBEM micrographs allows the identification of synapses with the same accuracy as in TEM micrographs.…”

“…In Drosophila , nc82 was shown to label amino acids 1,390–1,740 of BRP (Fouquet et al, 2009) and labeling was absent in BRP null mutants (Kittel et al, ). In locust brain homogenate, the antibody stained a double band at ∼220 kD in western blots (Leitinger et al, ); 2) AB5022 purchased from Chemicon, now Merck Millipore, a purified immunoglobulin from a polyclonal antiserum, raised in the rabbit against taurine‐glutaraldehyde. In the lamina neuropil of locust this antibody exclusively labeled compound eye photoreceptor cells (Leitinger, 2004), consistent with the staining pattern of another antibody in locust (Bicker, ).…”

“…First, we adapted the technique of serial block‐face scanning electron microscopy (SBEM) (Denk and Horstmann, ) to visualize, in 3D, the active zones of individual synapses between photoreceptor terminals and L cells. The presynaptic density, an electron‐dense aggregation of Bruchpilot‐reactive filaments to which vesicles attach before fusion, is the single most reliable indicator of the synaptic active zone (Leitinger et al, ). SBEM enables automated recording of scanning electron micrographs by repeatedly sectioning a tissue block, imaging the block surface each time a section is removed (Denk and Horstmann, ), enabling faithful 3D reconstructions of large datasets (Zankel et al, ).…”

“…SBEM is especially well suited for elucidating neuronal wiring (Briggman et al, ; Denk et al, ; Helmstaedter, ; Holcomb et al, ) but this is the first time it has been used to reconstruct synaptic connections in an invertebrate brain. Second, we used a marker for locust photoreceptors (Leitinger et al, ) and a marker for synaptic sites (Leitinger et al, ) to reveal the overall distribution of synapses over photoreceptor terminals in the lamina. This study has given us a novel evolutionary perspective on the design of visual processing for motion in insect eyes and reveals that tetrad synapses are not unique to flies and may have been around long before the first muscomorph flies appeared (Grimaldi and Engel, ).…”

Optimizing the 3D-reconstruction technique for serial block-face scanning electron microscopy

“…At first brains of the animals were dissected (see Leitinger et al, 2012). The left and right optic lobes were cut from the rest of the brain and fixed in 2.5% glutaraldehyde, 2% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4.…”