Developmental period for N-methyl-D-aspartate (NMDA) receptor-dependent synapse elimination correlated with visuotopic map refinement

Abstract

During a short perinatal interval, N-methyl-D-aspartate receptor (NMDAR) function is essential to a process in which spontaneous retinal waves focus retinal axon arbors in the superficial layers of the rodent superior colliculus (sSC). Here we provide evidence that this NMDAR-dependent axonal refinement occurs through elimination of uncorrelated retinal synapses arising from disparate loci, rather than stabilization of topographically appropriate inputs. The density of synaptic release sites within fluorescently labeled retinal terminals was counted in double-labeling experiments using confocal microscopy and antibodies against synaptophysin or synapsin-1. Chronic NMDAR blockade from birth increased retinal axon synapse density at postnatal days (P) 6, 8, and 10, suggesting that NMDAR currents reduce synapse density during the refinement period. With assay at P14, after focal arborization has been established, the effect disappeared. Conversely, chronic NMDA treatment, known to induce functional synaptic depression in the sSC, decreased retinocollicular synapse density at P14, but not earlier, during the refinement period (P8). Thus during the development of retinocollicular topographic order, there is a period when NMDAR activity predominantly eliminates retinal axon synapses. We were able to extend this period by using retinal lesions to reduce synaptic density in a defined zone. Synapse density on intact retinocollicular axons sprouting into this zone was increased by NMDAR blockade, even when examined at P14. Thus, the period of NMDAR-dependent synaptic destabilization is terminated by a factor related to the density and refinement of retinal arbors.

Figure 1. Antibodies used for immunohistochemistry recognize a single band in juvenile superior colliculus

Western blots from 4–14% poly-acrylamide gels after running denatured, whole lysate of the superficial visual layers of superior colliculus of P14 rats. The scans of lanes have been approximately aligned to show the relative kDa of bands. The lanes are as follows: (a) synaptophysin, (b) synapsin-1, (c) GAD 65/67, (d) GluR1, (e) GluR2. The GAD 65/67 antibody recognizes a doublet as expected, while the others recognize a single band as can be seen in the figure that includes the entire length of each lane.

Figure 2. Counting of retinal ganglion cell axon synapses

Synapses were identified by immunolabeling for synaptophysin combined with anterograde tracing of the contralateral retinal projection using fluorescently tagged cholera toxin. A. Sampling scheme. A confocal micrograph of the contralateral retinal projection (green) is shown inset into a schematic of the sampled field within the sSC. B. A typical optical section of a sampled sSC field. Contralateral retinal axons are green; synaptophysin labeling is red. Synaptic density counting was performed on a stack of such sections from a 4 µm-deep volume. The outlines of cell bodies and blood vessels are seen as distinct areas of low synapse density. C. Detail showing the distribution of axons and synaptophysin stain and the pattern of overlap (yellow). D. A binary image of C created with the threshold protocol used for the synaptic density analysis (see Methods). Puncta formed by the overlap of the label larger the 0.16µm² and completely engulfed by the axon (large arrowheads) were counted as synapses. Smaller puncta (small arrowheads) and partial overlap (asterisk) were not counted. Scale bars 20 µm, 10µm.

Figure 3. Immunohistochemistry for synapse associated proteins can be used to count retinal ganglion cell axon synapses

A. The pattern of synaptophysin staining within an axon is consistent with its identity as a synapse. The image is a collapsed confocal z-series from a 10 µm depth of tissue. Synaptophysin puncta occur in expansions of the axon either at terminal points or en passant. This image was taken within the stratum opticum of the ipsilateral colliculus where the axonal caliber is larger and synaptic density is lower. This allows for reconstruction over greater depths to visualize the entire arbor without interference from other axons. Other images presented in this paper are single confocal sections, which is required because of the density of contralateral axons in the SGS (for example, Fig. 2). A single confocal section from the contralateral retinal projection zone that contains a portion of arbor is shown in the inset. The localization of synaptophysin is similar to that seen in the ipsilateral projection, where the exact localization can be better ascertained. B. Sample confocal micrograph showing double labeling of retinal axons (green) and GAD 65/67 (red). Unlike the synaptophysin double labeling (Fig. 2B), there was no overlap between the two labels here. Even though our antibody recognizes GAD 65 and GAD 67, the majority of staining was restricted to processes with only light staining of cell bodies, indicating that GAD 65 was primarily localized during these overlap experiments. C. Correlation coefficients of overlap between ipsi axons and the immunostain for synaptophysin or GAD 65/67. The GAD stain is used as a control because it should never co-localize with retinal axons. Positive correlation indicates that the overlap of axon and antigen is greater than would be expected by chance (see Methods). This correlation coefficient was determined for the total number of overlapped “Pixels”, as well as for the number of pixel accumulations that exceeded 0.16 µm² (“Puncta”), our minimal criterion for a synapse. Synaptophysin antigens show more overlap, and GAD-65/67 localization shows less overlap, than would be expected by chance, especially when the minimal size criterion is imposed. Data for each antigen were acquired from 12 sections from two animals. D. Overlapped synaptophysin puncta are adjacent to post-synaptic markers. A confocal micrograph of tissue triple labeled for the ipsilateral RGC axons (green), synaptophysin (red – overlap is yellow), and also for the post-synaptic glutamate receptor GluR1 (blue). Presumptive synapses are marked with wide arrowheads. All identified presumptive synapses are adjacent to at least one GluR1 puncta that does not overlap with the retinal axon (thin arrowheads). Scale bars 10 µm.

Figure 4. NMDA receptor activity regulates synaptic density on contralateral retinal axons in an age dependent manner

Sample confocal micrographs of the SGS from various ages and treatment groups that show contralateral retinal axons (green) and synaptophysin label (red). Regions of overlap (yellow) that met the criteria for synapses are marked by a white circle. Drug treatment was provided by the slow release plastic Elvax in which an NMDAR antagonist (D-AP5), an agonist (NMDA) or an appropriate control (L-AP5 or water) was infused. Pups were killed during the period of retinotopic map refinement (P8) or after eye opening and map-refinement (P14). The panels are from the following treatment groups. A. P8 Control, four synapses; B. P8 NMDAR antagonist-treated, thirteen synapses; C. P8 NMDAR agonist-treated, four synapses; D. P14 Control, fifteen synapses; E. P14 NMDAR antagonist-treated, twelve synapses; F. P14 NMDAR agonist-treated, five synapses. Scale bar 10 µm.

Figure 5. NMDA receptor activity suppresses synapse formation during the period of retinocollicular map refinement

The average and standard error of RGC axon synaptic density for two experimental treatments--(1)NMDAR antagonist (D-AP5) treated (red) and (2) NMDAR agonist-treated (green)--and (3) the grouped controls are plotted. The control littermates from each of these experiments (L-AP5 and sham Elvax, respectively) have been grouped. NMDAR blockade increased synaptic density when animals were killed on P6, P8 or P10 but not P14. Conversely, agonist-treatment, known to functionally depress synapses, reduced densities at P14 but not P8. That NMDAR blockade increases synaptic density during the period of topographic refinement suggests that at this time the receptor is functioning to actively reduce synapse number on RGC axons. B. A second analysis of the same images to determine total density of synaptophysin staining within the retinal axons. This analysis shows our results are not dependent on the minimum size criterion for puncta, but that total synaptophysin density is regulated in a manner similar to synaptic density. Double asterisk indicates significant difference (p<0.01) from the age-matched, treatment-specific control group.

Figure 6. Decreased axonal density allows NMDAR blockade to increase synaptic density

A. A small lesion of the temporal retina deafferents a region of the rostral sSC. This is shown here in a whole mount of the mid and forebrain with cortex removed. The retinal projection from the lesioned eye has been labeled with a green fluorescent tracer. The scotoma can be seen in the whole mount by lack of green staining. Lesions were made at P6 and the animals sacrificed at P14. Under these conditions, axons from the remaining ganglion cells sprout into the deafferented region. B. Synaptic density, measured as described in Figure 1 and Figure 3 for control and NMDAR blocked (D-AP5) littermates, is increased by NMDAR blockade along the sprouting axons inside the deafferented region. C. Sprouted axons are of lower density, but can be examined for synapses as in the unlesioned projection. Shown here is a high power confocal micrograph taken within the deafferented region of a control pup in a manner the same as described in Figure 1. The retinal axons are green; synaptophysin stain is red. Synapses are marked with white circles. D. Similar image from within the deafferented region of an NMDAR antagonist treated pup. Synapses density is higher along the retinal axons. ** p < 0.01. Scale bars 1mm, 10 µm.