Spatial distribution of excitatory synapses on the dendrites of ganglion cells in the mouse retina

Abstract

Excitatory glutamatergic inputs from bipolar cells affect the physiological properties of ganglion cells in the mammalian retina. The spatial distribution of these excitatory synapses on the dendrites of retinal ganglion cells thus may shape their distinct functions. To visualize the spatial pattern of excitatory glutamatergic input into the ganglion cells in the mouse retina, particle-mediated gene transfer of plasmids expressing postsynaptic density 95-green fluorescent fusion protein (PSD95-GFP) was used to label the excitatory synapses. Despite wide variation in the size and morphology of the retinal ganglion cells, the expression of PSD95 puncta was found to follow two general rules. Firstly, the PSD95 puncta are regularly spaced, at 1-2 µm intervals, along the dendrites, whereby the presence of an excitatory synapse creates an exclusion zone that rules out the presence of other glutamatergic synaptic inputs. Secondly, the spatial distribution of PSD95 puncta on the dendrites of diverse retinal ganglion cells are similar in that the number of excitatory synapses appears to be less on primary dendrites and to increase to a plateau on higher branch order dendrites. These observations suggest that synaptogenesis is spatially regulated along the dendritic segments and that the number of synaptic contacts is relatively constant beyond the primary dendrites. Interestingly, we also found that the linear puncta density is slightly higher in large cells than in small cells. This may suggest that retinal ganglion cells with a large dendritic field tend to show an increased connectivity of excitatory synapses that makes up for their reduced dendrite density. Mapping the spatial distribution pattern of the excitatory synapses on retinal ganglion cells thus provides explicit structural information that is essential for our understanding of how excitatory glutamatergic inputs shape neuronal responses.

Figure 1. PSD95-GFP expression in the mouse RGC.

(A) The PSD95-GFP puncta distributed along the dendrites of a G6 ganglion cell. This montaged confocal image was assembled from three high-resolution images taken by a 100X oil-immersion objective. (B) Vertical projection of the same cell showing the depth of dendritic stratification in the IPL. (C) Magnified view of the area boxed in (A). A part of soma is visible at the left, and the synaptic zones labeled by PSD95-GFP are shown as puncta of high pixel intensity on the dendrites. INL, the inner nuclear layer; IPL, the inner plexiform layer; GCL, the ganglion cell layer. Scale bar, 20 µm in (A) and 10 µm in (C).

Figure 2. Synaptic input zones are uniformly distributed along the dendrites of most types of ganglion cells.

The NeuroLucida drawings in the upper panel are the dendritic morphologies of the G3, G5, G6, G7, G11, G12, G15, and G17 types of mouse RGCs characterized in the present study. The excitatory glutamatergic inputs (PSD95-GFP puncta) are indicated by red dots. Vertical projection of the same cells in the lower panel shows the depth of dendritic stratification in the inner plexiform layer (shaded area). Scale bar, 20 µm.

Figure 3. Spatial patterns of excitatory inputs are similar across different types of ganglion cells.

(A) The distribution of intervals between puncta. In the total distribution of intervals, it appears that all eight types of ganglion cells have their highest frequency at about 1–2 µm. The actual distribution of PSD95 puncta is compared with the distribution of the same number of puncta on that same dendritic structure at random (dotted lines). (B) The distribution of nearest-neighbor puncta distance. It is apparent that the excitatory synapses are excluded from a reference synapse site at the distance around 1–2 µm for all ganglion cell types. (C) The density of excitatory synaptic inputs as a function of dendritic branch order. In general, there are few synaptic inputs on the first several orders (or, the proximal zone of the dendritic field), and then the linear density of puncta gradually increases and reaches a plateau in the distal zone of the dendritic field in some but not all ganglion cell types. (D) The density of excitatory synaptic inputs as a function of distance from the soma (or, the Sholl analysis). For all cell types, the linear density of puncta near the soma is low. However, the puncta density, once outside the proximal zone, is relatively homogenous across the dendritic field in some but not all ganglion cell types. Note if more than one cell of a cell type has been sampled, the average value is plotted, but the error bar has been omitted for clarity.

Figure 4. Cells with a larger dendritic field receive excitatory synaptic inputs more efficiently than those with a smaller dendritic field.

(A) The plot of the PSD95 puncta numbers as a function of distance from the soma for eight different ganglion cell types. For clarity, four ganglion cell types with similar dendritic patterns (G3, G6, G7, and G11) were plotted in the upper panel, and the rest of cell types were in the lower panel. All cell types show a peak of puncta numbers at a location slightly displaced from the soma. (B) The plot of puncta number per membrane area as a function of distance from the soma for the same ganglion cells. There is a trend of increased puncta/membrane at distal dendrites. (C) The scatter plot of linear puncta density, dendritic arbor density (upper panel), and areal puncta density (lower panel) of each ganglion cell and its dendritic field diameter. Although the dendritic arbor density decreases when the dendritic field size increases, the linear puncta density is slightly higher for cells with a larger dendritic field size. Note if more than one cell of a cell type has been sampled, the average value is plotted, but the error bar has been omitted for clarity.