Journal: Frontiers in Neural Circuits

Article Title: Distribution of glycine receptors on the surface of the mature calyx of Held nerve terminal

doi: 10.3389/fncir.2014.00120

Figure Lengend Snippet: Analysis of membrane-localized GlyR alpha1-immunoreactivity in 3D reconstructed calyceal processes . (A) Three consecutive ultrathin sections through a calyceal swelling (CH) and a PC. The calyx is immunostained for vGluT1 (peroxidase) and for GlyR α1 (IG particles; indicated by black circles) along the presynaptic plasma membrane (outlined red). The postsynaptic density (PSD) of synaptic contacts between the calyx and PC are labeled green. (B) 3D alignment of digital contours of 29 serial sections (each 70 nm thick) of the same calyceal segment as in (A) , showing the dispersed distribution of presynaptic GlyRs (white dots) and synaptic contacts (green). (C) Electron micrograph showing clusters of GlyR-associated IG particles (arrowheads) at postsynaptic sites juxtaposed with putative inhibitory terminals (inh.). Scale bars: 0.5 µm (A,C) .

Article Snippet: We used polyclonal rabbit antibodies recognizing the second intracellular loop of the α1 subunit of GlyR (0.75 μg/ml) or vesicular GABA transporter (vGAT; 1.7 μg/ml), and polyclonal guinea pig antibodies, against vesicular glutamate transporter 1 (vGluT1; 1.25 μg/ml) (all from Synaptic Systems, Germany).

Techniques: Membrane, Clinical Proteomics, Labeling

Journal: Frontiers in Neural Circuits

Article Title: Distribution of glycine receptors on the surface of the mature calyx of Held nerve terminal

doi: 10.3389/fncir.2014.00120

Figure Lengend Snippet: Localization of GlyRs in identified calyceal processes . (A,A’) Two consecutive sections through a calyceal swelling (CH) double labeled with antibodies against vGluT1 (peroxidase) and GlyR α1 (IG particles; arrows). Asterisks denote synaptic contacts between the calyx and a PC. White arrowheads in panel A indicate borders between membrane regions that are either connected or unconnected to the soma of a PC. (B) A semitransparent superposition of 29 contoured sections as in (A) to reconstruct a segment of swelling with several synaptic junctions (green areas) and numerous GlyRs (red dots). (B’) The same stack of contoured sections as in (B) viewed from a different angle (the PC facing side). Black and red dots illustrate GlyRs localized in the calyceal membrane that is either connected or unconnected to the soma of a PC. (C) EM image of anti-vGluT1 labeled calyceal stalk immunoreactive for GlyR α1. Sectors in boxes (a–d) , displayed on an expanded scale below, show examples of GlyRs (arrows) on plasma membrane that is unconnected (a,d) to the postsynaptic cell or that encloses extended extracellular spaces (b,c) . (D) The 3D reconstruction of 28 serial sections as in (C) illustrating a semitransparent segment of calyceal stalk forming synaptic contacts (green areas) with a PC and containing numerous GlyRs (red dots). (D’) The same stack of contours as in (D) viewed from a different angle (the PC facing side). Scale bar: 1 µm (A’,C) .

Article Snippet: We used polyclonal rabbit antibodies recognizing the second intracellular loop of the α1 subunit of GlyR (0.75 μg/ml) or vesicular GABA transporter (vGAT; 1.7 μg/ml), and polyclonal guinea pig antibodies, against vesicular glutamate transporter 1 (vGluT1; 1.25 μg/ml) (all from Synaptic Systems, Germany).

Techniques: Labeling, Membrane, Clinical Proteomics

Journal: Frontiers in Neural Circuits

Article Title: Distribution of glycine receptors on the surface of the mature calyx of Held nerve terminal

doi: 10.3389/fncir.2014.00120

Figure Lengend Snippet: GlyRs tend to accumulate in calyceal swellings proximal to inhibitory boutons. (A,A’) Two consecutive sections through a calyceal swelling (CH), immunoreactive for vGluT1 (peroxidase) and GlyR α1 (IG particles; arrows), and vGluT1-immunonegative, putatively inhibitory bouton (inh.). Asterisks denote synaptic contacts between the bouton and a PC. Inset shows an example of a flat vesicle (arrow) at the inhibitory AZ and a cluster of IG particles at the inhibitory synaptic contact. Scale bar: 0.5 µm. (B) Stack of contours from 17 serial EM images of sections shown in (A) . Note an accumulation of GlyRs (red dots) in calyceal compartments adjacent to the bouton. Synaptic contacts made by the calyx or by the bouton with a PC are shown in green or blue. (C) Cumulative histogram showing the distribution of spatial distances between each IG particle (solid line) on the calyceal segment shown in (B) and the middle of the PSD of the nearest synaptic contact between the inhibitory bouton and a PC (see inset in panel D ). For comparison, the distribution of the distances between digitized points delineating the surface of the swelling and the middle of the PSD is shown (dotted line). The distributions significantly differ ( P < 0.001; Kolmogorov-Smirnov test; bin width = 0.2 µm). (D) The distribution of distances provided by similar measurements as in (C) . The data was collected from 12 segments of swellings adjacent to an inhibitory bouton. The lengths are plotted relative to the longest distance in a segment. The distributions of IG particles and surface points are significantly different ( P < 0.001; Kolmogorov-Smirnov test; bin width = 0.05). (E) The histogram shows spatial distances between each IG particle (solid line) or each digitized surface point (dotted line) of a calyceal segment and a reference point (P) which was arbitrarily set at the horizontal edge of a section that is placed in the middle of the segment (see inset).The data was collected from 21 swellings without any detectable inhibitory bouton in their proximity. Note that the distribution of IG particles in these segments is not random ( P < 0.001; Kolmogorov-Smirnov test; bin width = 0.05). (F,G) The plots show relative frequencies of IG particles in five partitions of perimeters of sections constituting segments of swellings with ( F ; N = 14) or without ( G ; N = 37) an inhibitory bouton in their proximity. Each data point represents the sum of IG particles found in one of the partitions of sections constituting a segment (see schemes in insets). Mean values are indicated by horizontal bars. The quantity of IG particles in each partition was normalized to the total number of the particles in a segment. The partitions are labeled 1–5 while that containing a reference point (blue in each inset) is the partition #1. The reference point was set either as an intersection of the calyceal membrane and a link between midpoints of cross-sections through a swelling and a bouton (F) or at the horizontal edge of a swelling (G) . Note significantly increased amounts of IG particles in the partition adjacent to the inhibitory bouton. *** P < 0.001; ** P < 0.01; * P < 0.05; Dunnett’s multiple comparison test (data from the partition #1 were used as a control).

Article Snippet: We used polyclonal rabbit antibodies recognizing the second intracellular loop of the α1 subunit of GlyR (0.75 μg/ml) or vesicular GABA transporter (vGAT; 1.7 μg/ml), and polyclonal guinea pig antibodies, against vesicular glutamate transporter 1 (vGluT1; 1.25 μg/ml) (all from Synaptic Systems, Germany).

Techniques: Comparison, Labeling, Membrane, Control