Chronic NMDA receptor blockade from birth increases the sprouting capacity of ipsilateral retinocollicular axons without disrupting their early segregation

Abstract

We have investigated the role of the NMDA glutamate receptor (NMDAR) in the genesis and regulation of structural plasticity during synaptogenesis in the visual layers of the rat superior colliculus (sSC). In this neuropil, three projections compete for synaptic space during development. By fluorescently labeling the projections of both eyes and imaging them with confocal microscopy, we can quantify the sprouting of the ipsilateral retinal projection that follows removal of a portion of the contralateral retinal and/or corticocollicular projection. Using these techniques we have studied the effects of NMDAR blockade under different levels of competition. NMDARs were chronically blocked from birth [postnatal day 0 (P0)] by suspending the competitive antagonist 2-amino-5-phosphonopentanoic acid in the slow release plastic Elvax, a slab of which was implanted over the sSC. Such treatment alone does not impair the normal segregation of the retinal projections. However, if sprouting of the ipsilateral projection is initiated with a small contralateral retinal lesion at P6, this sprouting can be further increased by blocking NMDARs from birth. Sprouting of the ipsilateral retinal projection is also induced by retinal lesions made at P10/P11, but NMDAR blockade does not augment the sprouting induced by this later lesion. However, when combined with simultaneous ablation of the ipsilateral visual cortex, P10/P11 lesions show increased sprouting after NMDAR blockade. These data indicate that P0 NMDAR blockade does not eliminate synaptic competition in the sSC. Instead, early elimination of NMDAR function appears to facilitate sprouting that is gated in a stepwise manner by the other visual afferents.

Fig. 1.

Simultaneous labeling of eyes with fluorophore-conjugated CTB allows detailed imaging of both retinocollicular projections. A, Flat mount of a P14 retina that has been injected with FITC CTB. Retinal ganglion cell axons stream to the optic disk evenly from all quadrants. B, C, Montages of confocal micrographs from a single hemisphere from the rostral region of a P14 rat sSC cut in coronal section. Each section is a brightest pixel projection of a 20-μm-deepz series. The contralateral (B) and ipsilateral (C) retinal afferents have been labeled by intraocular injection of CTB conjugated to tRITC or FITC, respectively, and were imaged simultaneously through two channels. The contralateral projection forms a dense sheet in the SGS and SZ (B), whereas the ipsilateral projection is mostly confined to the SO, with denser SGS projections n the medial and lateral edges (C). Refined arbor patches can be seen (arrows) at this age. SZ, SGS, and SO refer to the layers of the visual sSC. D, Detail ofC showing the structure of an ipsilateral arbor. Label is dominant in the arbors (asterisk) although the axons are still visible (arrow). Scale bars: A, 1 mm; B, C, 200 μm; D, 100 μm.

Fig. 2.

NMDAR blockade from birth does not perturb the refinement of the retinocollicular projection. A, B, Montages from a P14 sSC section that has been treated from birth with AP5-Elvax. The section is from a rostrocaudal locale similar to that in Figure 1. Both the contralateral (A) and ipsilateral (B) projections appear normal. In particular, the ipsilateral retinal ganglion cell axons have become normally restricted to the SGS and have refined to form the patchy arborizations (arrow) characteristic of a refined ipsilateral retinal projection. C, Quantification of the density of the retinocollicular projection to the ipsilateral sSC from the pups of a single litter that had received either no treatment (control) or chronic treatment with the inactive stereoisomer (l-AP5) or the racemic mixture (AP5) of the competitive NMDA receptor antagonist and were killed at P14. The density of the ipsilateral retinal axon in two-dimensions is represented as the labeled pixel density (see Materials and Methods). These densities were measured in micrographs at least 500 μm from the medial or lateral edges using 12 sections taken from three pups of each group. The sections were alternate 100 μm sections beginning 400 μm from the rostral edge. This sampling pattern was identical to that used to quantify the densities after a lesion. There are no significant differences among any groups (ANOVA). Scale bar: A, B, 200 μm.

Fig. 3.

NMDAR blockade does not perturb the refinement of the ipsilateral retinal projection to the sSC at P6. A, B, Confocal micrographs from P6 pups chronically treated withl-AP5 (A) or AP5 (B). Only the ipsilateral retinal ganglion cell axons are labeled. Theframes are from the lateral half of the rostral sSC. At this early age most of the axons are already confined to the upper SO; few axons extend into the SGS. C, Quantification of ipsilateral arbor density in the rostral SGS, at least 300 μm from the medial or lateral edges (n = 18 sections from 6 pups for the l-AP5 treatment and 15 sections from 5 pups for the AP5 treatment). Scale bar: A, B, 100 μm.

Fig. 4.

Small retinal lesions made at P6 or P10/P11 cause a scotoma in the contralateral sSC that permits sprouting of the ipsilateral retinal ganglion cell axons. A, Microcautery lesions destroy a small, defined section of retina.Left, The temporal portion of a thin, frontal section of a lesioned retina, 8 d after the lesion, shows the area of the lesion (arrowheads) with healed sclera surrounding it. The ganglion cell layer is labeled bright red by the CTB. Right, Whole mount of another lesioned retina (8 d after the lesion) shows the ablated area in the temporal pole (arrowheads). There is a reduction in the density of bundled axons entering the optic disk from the lesioned region.B, Top view of the dorsal diencephalon and midbrain shows the primary targets of retinal ganglion cells, the sSC (sc) and dorsal lateral geniculate nucleus (lgn). One eye has been labeled with FITC CTB (green) and the other with tRITC CTB (red); this latter eye also received a lesion in the superiotemporal pole of the retina at P6. This lesion causes a scotoma (surrounded by arrowheads) in the laterorostral pole of the contralateral sSC and caudal dorsal LGN 8 d later.C, D, Confocal montages are shown of 20 μmz-series projections taken from coronal sections from the locations marked c and d inB. These show sprouting of ipsilateral retinal ganglion cells axons into the SGS and particularly the SZ within the scotoma (arrowheads). E, F, Singleframes from the areas shown in D(e, f) illustrate the pattern of sprouting in the SGS and SZ (delineated by the dashed line).G, Quantification is shown of ipsilateral axon density within the SGS/SZ inside the scotoma from two litters (onered, the other blue) in which half of the pups received P6 lesions (triangles) and half of the pups received P11 lesions (squares). All animals were killed 8 d after the lesion. Each point is the average labeled pixel density (see Materials and Methods) for a single animal derived from three sections. Within each litter P6 lesions cause more plasticity than do P11 lesions, but the variability between litters is as large as the difference between early and late lesions within a single litter. Two-way ANOVA indicated significant differences between litters (p < 0.0001) and lesion time (p < 0.0001). Within litters there is was no relationship between size of the lesion and plasticity.D, Dorsal; V, ventral. Scale bar:A, B, 1 mm; C, D, 200 μm; E, F, 100 μm.

Fig. 5.

Eight days after the lesion is sufficient to restore normal synaptic density in the SGS. Confocal micrographs of immunohistochemical staining for synaptophysin reveal punctate, presumably synaptic, staining. A, B, Two days after a P11 lesion the puncta are reduced inside the scotoma (B) compared with outside the scotoma (A) in the same section. C, D, However, 8 d after a P11 lesion there is no observable difference inside (D) and outside (C) the scotoma. Scale bar, 10 μm.

Fig. 6.

NMDA receptor blockade from birth increases the sprouting in response to a P6 retinal lesion. Confocal micrographs of a 20 μm z-series projection show a greater density of ipsilateral retinal ganglion cell axons within (B, C, E, F), but not outside (A, D), the scotoma when sections from pups treated with l-AP5 (A–C) are compared with those treated with AP5 (D–E). B and Eshow a characteristic response in the crown of the rostral sSC, whereasC and F show a more lateral region. Scale bar, 100 μm.

Fig. 7.

Quantification of the increased ipsilateral retinal axon sprouting in the SGS/SZ in response to a P6 retinal lesion after chronic NMDAR blockade. The amount of sprouting into the scotoma is measured in each section by subtracting the labeled pixel density (see Materials and Methods) of a frame outside the scotoma from that of a frame inside the scotoma. To control interlitter variability, this increased labeled pixel density is normalized to the average response of the l-AP5 pups from each litter. The data were gathered from two litters, each divided into l-AP5-Elvax- and AP5-Elvax-treated groups. From each pup increased labeled pixel densities were measured from three sections in the rostral region of the scotoma (see Materials and Methods). Double asterisks indicate p < 0.01 byt test.

Fig. 8.

The occipital cortex inhibits ipsilateral retinal sprouting in response to a P10/P11 retinal lesion, and its removal allows NMDAR blockade to augment this sprouting. A, NMDAR blockade from birth does not affect the sprouting in response to a P10 retinal lesion.A₁–A₄, Confocal micrographs of a 20 μm z-series projection show that both l-AP5 (A₁, A₂)- andd-AP5 (A₃, A₄)-treated pups have similar ipsilateral retinal axon densities within (A₂, A₄) and outside (A₁, A₃) the scotoma. A₅, Quantification is as described in Figure 7. B, Removal of the ipsilateral occipital cortex simultaneous with lesion of the contralateral retina causes more sprouting than does the retinal lesion alone.B₁–B₄, Micrographs are from pups that received a small retinal lesion at P10/P11 (B₁, B₂) or retinal lesion and simultaneous aspiration of the occipital cortex (B₃, B₄). B₂ andB₄ are from inside the scotoma and show elevated sprouting, particularly in the upper SGS, that is caused by cortex lesion. Another pattern of elevated sprouting, in this case in the lower and middle SGS, is shown in thel-AP5-treated sSC ofC₂. B₁ andB₃ are from outside the scotoma where the cortical lesion also caused an elevation in ipsilateral sprouting (see Fig. 9). B₅, Quantification of ipsilateral retinal axon plasticity is as described in Figure 7, but litters are normalized to control pups with a retinal lesion alone.Double asterisks indicate p < 0.01.C, Removal of the occipital cortex allows NMDAR blockade to increase sprouting further.C₁–C₄, Micrographs are from pups receiving simultaneous retinal and occipital cortex lesions at P10/P11 and being treated with the inactive isomerl-AP5 (C₁, C₂) or the active isomer AP5 (C₃, C₄). There is more sprouting in the scotoma (C₂, C₄) but similar density outside (C₁, C₃) after NMDAR blockade.C₅, Quantification of ipsilateral retinal plasticity is as described in Figure 7. Note that because thel-AP5-Elvax-treated sSCs with retinal and cortical lesions are now the control, their normalized increased labeled pixel density is now 1; this does not imply thatl-AP5 treatment reduces the plasticity to dual lesion by one-third but is a function of the way in which the numbers were normalized. A single asterisk indicatesp < 0.05. Scale bar, 100 μm.

Fig. 9.

Ipsilateral retinal ganglion cell axon density outside the scotoma is increased by removal of the occipital cortex but not by NMDA receptor blockade from birth (Fig. 8, compareodd-numbered frames). Labeled pixel intensities were calculated for three frames outside the scotoma (Fig.4D, box e) from three sections of each pup from the litters used in the previous experiments. Normal andl-AP5-Elvax treatment groups showed no differences and were grouped for this comparison. Single and double asterisks represent p < 0.05 andp < 0.01 differences from control, respectively, by Tukey pairwise post hoccomparison.