Sampling and methodological controls
We focused our analysis on ventral (v) and dorsal (d) midline subregions of the mid DR () because they contain the highest density of 5-HT neurons (Steinbusch, 1984
; VanderHorst and Ulfhake, 2006
). Array tomography allows examination of a broader XY area in comparison to electron microscopy; therefore, in this study both areas of the DR quantified were contained in the same block and sections. The density of TPOH labeled structures was comparable in these two areas and accounted for about 14% of the total volume. Examination of tissue sections processed for immunolabeling without primary antisera or for elution of immunolabeling revealed negligible fluorescence labeling.
In order to control for possible changes in antigenicity due to several rounds of staining/elution, we included immunolabeling for TPOH and PSD-95 in each round. Analysis of the reproducibility of immunolabeling with these antigens between different rounds of staining/elution showed a very similar pattern of immunolabeling for PSD-95 and TPOH, indicating that the antigenicity is substantially preserved at least after three rounds (). That is, a high proportion of the same pixels were relabeled after consecutive rounds of staining/elution for TPOH (r = 0.94) and PSD-95 (r = 0.89) (). Remaining pixels that were not repeatedly detected either represent nonspecific immunolabeling or specific immunolabeling that falls below the level of detection in two of three rounds. In addition, the total amount of PSD-95-immunolabeled puncta either associated or not with TPOH-immunolabeled processes was also similar for the same stack of images among different rounds of staining/elution ().
Volumetric images rendered from images of serial sections illustrate the high-resolution achieved in array tomography (). Therefore, individual synaptic elements (e.g., PSD-95) and their associations with TPOH-immunolabeled processes can be easily detected and subjected to quantitative automated analysis.
Figure 3 Volumetric image of labeling for TPOH and PSD-95 in the mouse DR, illustrating the high-resolution achieved in array tomography. A: A volume rendering of 30 ultrathin (70 nm) serial sections showing a high density of TPOH-positive cells (green) abundantly (more ...)
Glutamatergic axon terminals within the DR
Qualitative examination of the patterns of immunolabeling revealed that immunolabeling for all three types of VGLUT yielded punctate labeling widespread in both subregions of the DR studied (), consistent with a primary localization within axons. In general, puncta immunolabeled for one type of VGLUT did not contain labeling for either of the other two. Also, immunolabeling for each VGLUT often appeared at the same location through several serial sections (), although there were instances where labeling appeared only in single sections.
Figure 4 All three types of VGLUT and PSD-95 are widely distributed in the mouse DR. A: An image of a single 70-nm section showing a high density of TPOH-positive cells (white) within the DR, densely surrounded by PSD-95 (red), VGLUT1 (green), VGLUT2 (magenta), (more ...)
Figure 5 Quantitative analysis of PSD-95 and VGLUT puncta in the mouse DR. A: Density of PSD-95 puncta, and their association with 5-HT cells in the ventral (vDR) and dorsal (dDR) parts of the DR. B–D: Serial section images through individual VGLUT1 (green), (more ...)
We completed two different types of analyses to quantify the distribution of VGLUT immunolabeling. The first one involved a broad and less conservative approach of quantifying all individual immunolabeled elements within volumetric images in total and in association with TPOH immunolabeling. The second approach was a more conservative analysis where a contingency was imposed to exclude biologically less relevant and nonspecific immunolabeling. In a subset of the data we analyzed VGLUT-immunolabeled puncta that colocalized with synapsin I. Subsequently, we did a comprehensive analysis of VGLUT-positive elements that had a relationship to the postsynaptic protein PSD-95. We focused on the PSD-95 analysis with the aim of getting specific insight into instances of VGLUT immunolabeling that corresponded to potential synapses.
The first analysis of all instances of VGLUT immunolabeling showed a similar regional distribution of each type of VGLUT in both the dorsal and ventral portions of the midline DR, as there were no significant interaction between subregion and VGLUT type (F2,12 = 0.75, P = 0.492) or a main effect of subregion (F1,12 = 1.37, P = 0.264) (). However, we found differences in the density of each VGLUT type within both studied subregions (F2,12 = 11.38, P < 0.002). Specifically, we found that there was a greater density of VGLUT2-immunolabeled puncta than VGLUT1 puncta overall in the DR (P < 0.002), and nonsignificantly different from VGLUT3 (P = 0.06) ().
In order to get a measure of the potential interactions between puncta immunolabeled for each of VGLUTs and 5-HT cells, we quantified instances where VGLUT-immunolabeled objects overlapped with TPOH-labeled profiles. This accounted for 42% of the total number of VGLUT1-3-immunolabeled puncta in the ventral DR and 35% in the dorsal DR. We did not find a significant interaction effect between subregion and VGLUT type (F2,12 = 0.10, P = 0.904) or a selective association with one type of VGLUT versus another (F2,12 = 2.45, P = 0.128). Also, the total amount of VGLUT1-3 puncta associated with 5-HT neurons was not significantly different between both subregions (F1,12 = 0.95, P = 0.350) ().
Colocalization of VGLUTs with synapsin I
This first analysis of total VGLUT-immunolabeled puncta is likely an overestimation of axon terminals because both smaller objects and potentially nonspecific labeling may be included. We further examined whether these VGLUT-positive puncta might also be related to a general marker for presynaptic axon terminals such as synapsin. Although this protein is thought to be one of the best general markers for synaptic boutons, it may be present at different levels in different populations of axon terminals and/or at low levels. Therefore, colocalization with synapsin would provide a conservative estimate of synaptic boutons (Micheva et al., 2010a
). For this analysis we processed two of the volumes sampled containing 29 sections including both dorsal and ventral subregions of the DR. We found that 26%, 34%, and 30% of VGLUT1-, VGLUT2-, and VGLUT3-immunolabeled objects, respectively, were dually labeled for synapsin, confirming that at least this subset of VGLUT-labeled puncta correspond to accumulations of synaptic vesicles. Remaining puncta could be accounted for among various possible groups including synaptic boutons with subthreshold levels of synapsin, a biologically relevant accumulation of VGLUT protein that is not associated with significant synapsin, and/or non-specific immunolabeling.
Apposition of glutamate axon boutons and PSD-95
In order to specifically study synaptic associations, we subsequently analyzed the density of VGLUT1-3-immunolabeled puncta in close apposition with the postsynaptic protein PSD-95 (). First, we found that PSD-95 independently was highly abundant and equally distributed in both studied subregions of the DR (F1,12 = 0.24, P = 0.636) (, ). PSD-95 puncta could be detected through several consecutive sections (), and was visible on the perimeter of TPOH-labeled processes and cell bodies. In addition, it was noted that some PSD-95 puncta were not on the surface of TPOH dendrites, but rather appeared within what is likely the cytoplasmic compartment of TPOH-immunolabeled profiles, possibly corresponding to components of the postsynaptic densities in transit to or from synaptic sites. A large population (≈65%) of PSD-95 puncta was not associated with any of the VGLUT presynaptic partners. In addition, in both studied subregions a substantial proportion (≈70%) of PSD-95 was not associated with 5-HT neurons, indicating likely association with non-5-HT cell processes ().
Figure 6 Quantitative analysis of VGLUT/PSD-95 puncta appositions in the mouse DR. A,B: Density of VGLUT/PSD-95 puncta appositions, and their association with 5-HT cells in the ventral (vDR) and dorsal (dDR) parts of the DR, respectively. C–E: Serial section (more ...)
Only a fraction of the total number VGLUT-immunolabeled elements was found in close apposition to PSD-95. 29% of VGLUT2 puncta were associated with PSD-95 while lower percentages, 20%, and 14% of VGLUT1 and VGLUT3 puncta, respectively, were associated with PSD-95. The higher rate of association of VGLUT2 with PSD-95, coupled with the overall abundance of VGLUT2 puncta, resulted in a higher absolute density of VGLUT2/PSD-95 pairs than VGLUT1/PSD-95 or VGLUT3/PSD-95 pairs () (F2,12 = 57.03, P < 10−4). Depending on the subregion and VGLUT type, VGLUT2/PSD-95 pairs predominated by 1.8–2.5-fold. Consistent with the higher density of VGLUT2/PSD-95 pairs, cross-correlation analysis between PSD-95 and VGLUT-immunolabeled pixels showed a higher degree of colocalization for VGLUT2 puncta with PSD-95 in comparison to VGLUT1 or VGLUT3 (). We also performed, as a control, cross-correlation analysis between VGLUT1 and VGLUT2 puncta, and found no evidence for a relationship (). Thus, all three VGLUTs specifically associate with PSD-95, and VGLUT2 predominates in this relationship.
We then evaluated the triple association between VGLUT/PSD-95 and 5-HT cells (). Only a proportion of the total number of VGLUT/PSD-95 pairs were associated with TPOH-immunolabeled profiles: 28%, 23%, and 32% for VGLUT1, 2, and 3, respectively. Thus, VGLUT2/PSD-95 pairs were somewhat underrepresented in the relationship to TPOH, and lost their consistent 2-fold predominance over VGLUT1 and VGLUT3. However, there were still significant differences in abundance of appositions containing each type of VGLUT in the vDR (F2,6 = 9.31, P < 0.01) and the dDR (F2,6 = 12.21, P < 0.01). In the vDR, VGLUT2/PSD-95 pairs in association with TPOH were 33% more numerous than those containing VGLUT3 (P < 0.02) and 25% more than those containing VGLUT1, although in this case statistical significance was not reached (P = 0.06) (). In the dDR, the number of VGLUT2-containing appositions associated to 5-HT cells were 2-fold more numerous than those containing VGLUT1 (P < 0.01) and 22% more than VGLUT3 (P < 0.05).
To summarize the differences in the synaptic arrangements in vDR versus dDR, there was a greater density overall of VGLUT/PSD-95 pairs in the vDR in comparison to dDR (F1,12 = 12.79, P < 0.004). In addition, VGLUT1-PSD-95 pairs were more commonly associated with TPOH cells in the vDR in comparison to the dDR (F1,4 = 39.30, P < 0.003).