Skip to main content
. Author manuscript; available in PMC: 2017 Jun 15.
Published in final edited form as: J Comp Neurol. 2016 Jan 4;524(9):1892–1919. doi: 10.1002/cne.23946

Figure 4. Normal distribution of VGLUT1 and VAChT contacts on calbindin-IR Renshaw cells.

Figure 4

A1, A2, A3, Triple immunofluorescence for calbindin (magenta, Cy3), VGLUT1 (green, FITC) and VAChT (white, Cy5). The images are 2D projections of 4 optical sections (obtained at 20×; 6 μm depth) and show all three immunoreactivities in different combinations. The location of three RCs reconstructed in following panels is indicated in A1 and with asterisks in A2. White dashed lines indicate the edge of the spinal cord gray matter and the boundary between lamina IX and VII. Green dashed line indicates the region with high density of VGLUT1-IR puncta. The dashed grey line indicates the region with the highest concentration of motor axon-derived VAChT-IR synaptic boutons. Each of the three RCs are located in different positions with respect to regions with high density VGLUT1-IR or VAChT-IR boutons. B1, Full reconstruction of RC#2 superimposed on a single optical section (through the RC2 cell body) from a high magnification (60×) tiled image. Contacts from VAChT-IR and VGLUT1-IR boutons are indicated as grey and green circles, respectively. Dashed line is the ventral border of the spinal cord gray matter. B2, Ninety degree rotation of RC#2 showing the section thickness. Dendrites oriented in the longitudinal rostro-caudal axis intersect the section surface faster than dendrites in more or less transverse orientations. Some transversally oriented dendrites reach their natural terminations within the section and usually end with small bi- or trifurcations (see asterisk in B1, B2 and also C). C, Reconstructions of three RCs with VGLUT1-IR (green dots) and VACHT-IR boutons (grey dots) mapped on their dendrites. VAChT-IR contacts are denser on dendrite segments coursing through the VAChT area, while VGLUT1-IR contacts are more frequent in dendrites entering the VGLUT1 area. D, Full reconstruction of RC#2 within Sholl circles. Two dendritic areas are boxed; one is in the first 50 μm Sholl (proximal) from the cell body center and the other at 200-250 Sholl distance (distal). E1, E2, E3 and F1, F2, F3 are Imaris surface renderings of these dendritic segments in three different rotations. Arrowheads indicate different views of the same contacts. The proximal segment (E) has a high density of VAChT-IR contacts, while the distal segment (F) receives predominantly VGLUT1-IR contacts. Scale bars: 100 μm in A1, 50 μm in B1 and 10 μm in E3 (F panels are at the same magnification as E).