Rearrangement of retinogeniculate projection patterns after eye-specific segregation in mice

Abstract

It has been of interest whether and when the rearrangement of neuronal circuits can be induced after projection patterns are formed during development. Earlier studies using cats reported that the rearrangement of retinogeniculate projections could be induced even after eye-specific segregation has occurred, but detailed and quantitative characterization of this rearrangement has been lacking. Here we delineate the structural changes of retinogeniculate projections in the C57BL/6 mouse in response to monocular enucleation (ME) after eye-specific segregation. When ME was performed after eye-specific segregation, rearrangement of retinogeniculate axons in the dorsal lateral geniculate nucleus (dLGN) was observed within 5 days. Although this rearrangement was observed both along the dorsomedial-ventrolateral and outer-inner axes in the dLGN, it occurred more rapidly along the outer-inner axis. We also examined the critical period for this rearrangement and found that the rearrangement became almost absent by the beginning of the critical period for ocular dominance plasticity in the primary visual cortex. Taken together, our findings serve as a framework for the assessment of phenotypes of genetically altered mouse strains as well as provide insights into the mechanisms underlying the rearrangement of retinogeniculate projections.

Figure 1. Effects of ME after eye-specific segregation on the remaining retinogeniculate projections in the mouse dLGN.

(A) A schematic of experimental procedures. ME was performed at P10, and CTB was injected into the other eye to visualize the remaining RGC axons. Contra and ipsi indicate contralateral and ipsilateral sides to the CTB-injected eye, respectively. The locations of the gap and the ipsilateral patch are also shown. (B–G) Coronal sections of 50 µm thickness were prepared from control (B, D, F) and ME-treated (C, E, G) mice between P35–P37. Representative images of the rostral (B, C), middle (D, E), and caudal (F, G) dLGN are shown. White lines show the boundaries of the dLGN. In each panel, the top is dorsomedial. Scale bar represents 200 µm.

Figure 2. Quantification of the effects of ME after eye-specific segregation on the remaining retinogeniculate projections.

ME was performed at P10, and CTB was injected into the other eye. Coronal sections were prepared from ME-treated and control mice between P35–P37. (A) The sizes of the CTB-positive areas relative to those of the dLGNs. The dLGNs ipsilateral to the CTB-injected eye were used. The CTB-positive area was significantly larger in ME-treated mice (n = 5) than that in control mice (n = 3). (**) P<0.01, unpaired Student's t-test. Error bars represent S.D. (B) The sizes of the gap relative to those of the dLGNs. The gap was significantly smaller in ME-treated mice (n = 4) than that in control mice (n = 4). (*) P<0.05, unpaired Welch's t-test. Error bars represent S.D. (C) CTB signal intensities within the dLGN contralateral to the CTB-injected eye. CTB signal intensities were plotted against the distance from the dorsomedial tip of the dLGN (see also Figure S2). The signal intensities in the area corresponding to the gap were higher in ME-treated mice (red) than in control mice (blue). Dark lines and light lines represent the averages and data derived from individual dLGNs, respectively.

Figure 3. Time course of the rearrangement of retinogeniculate projections induced by ME at P10.

ME was performed at P10, and CTB was injected into the other eye. Coronal sections through the dLGN ipsilateral to the CTB-injected eye were prepared at the indicated time points. The sizes of the CTB-positive areas relative to those of the dLGNs are shown. The CTB-positive areas were significantly larger in ME-treated mice than those in age-matched control mice within 2 days after ME. (**) P<0.01, (*) P<0.05, unpaired Student's t-test. Error bars represent S.D. The numbers in parentheses indicate the number of animals.

Figure 4. Critical period for ME-induced rearrangement of retinogeniculate projections.

ME was performed at the indicated time points, and CTB was injected into the other eye. Coronal sections through the dLGN ipsilateral to the CTB-injected eye were prepared 25–27 days later. The sizes of the CTB-positive areas relative to those of the dLGNs are shown. Note that the CTB-positive areas were significantly larger in mice treated with ME at P10 or at P22 compared with those in control mice. (**) P<0.01, (*) P<0.05, unpaired Student's t-test. The CTB-positive areas did not show significant differences when ME was performed at P34 (P = 0.91, unpaired Welch's t-test). These results suggest that the critical period for ME-induced rearrangement ends between P22 and P34. Error bars represent S.D. The numbers in parentheses indicate the number of animals.

Figure 5. ME-induced rearrangement of retinogeniculate projections along the O-I and DM-VL axes.

ME was performed at P10, and CTB was injected into the other eye. Coronal sections through the dLGN ipsilateral to the CTB-injected eye were prepared at the indicated time points, and the ipsilateral patches (gray area) were extracted from the CTB-positive areas. (A) A diagram of the dLGN showing the O-I and DM-VL axes. The lengths of the ipsilateral patch along the DM-VL axis (dark blue arrow) and the O-I axis (dark red arrow) were divided by the lengths of the dLGN along the DM-VL axis (light blue line) and the O-I axis (light red line), respectively (see Figure S5 for details). The values are shown in panels B and C. (B) The lengths of the ipsilateral patch along the O-I axis were significantly larger in ME-treated mice than in age-matched control mice at P15. Note that the lengths did not show significant differences between P15 and P25–P26 in ME-treated mice, suggesting that the expansion reached its maximum within 5 days. (**) P<0.01, (*) P<0.05, (N.S.) not significantly different (P≥0.05), unpaired Student's t-test for comparisons between ME-treated and control mice, and Tukey-Kramer test for comparisons between different ages within either the ME-treated group or the control mouse group. Error bars represent S.D. The numbers in parentheses indicate the number of animals. (C) The lengths of the ipsilateral patch along the DM-VL axis were significantly larger in ME-treated mice than in age-matched control mice. In contrast to the lengths along the O-I axis, the length along the DM-VL axis in ME-treated mice at P25–P26 was significantly larger than that at P15. (**) P<0.01, (*) P<0.05, (N.S.) not significantly different (P≥0.05), unpaired Student's t-test for comparisons between ME-treated and control mice, and Tukey-Kramer test for comparisons between different ages within either the ME-treated group or the control mouse group. Error bars represent S.D. The numbers in parentheses indicate the number of animals. These results suggest that the expansion of the ipsilateral patch along the DM-VL axis is relatively slow compared with that along the O-I axis.

Figure 6. The effects of MD and TTX on retinogeniculate projections.

(A) The effect of MD on retinogeniculate projections. P10 pups were treated with or without either ME, MD or anesthesia alone (sham). CTB was injected into the intact eye, and coronal sections of the dLGN ipsilateral to the CTB-injected eye were prepared at P35–P37. The sizes of the CTB-positive areas relative to those of the dLGNs were measured. In contrast to ME, MD did not significantly affect the size of the CTB-positive area. The data of control and ME-treated animals are the same as those in Figure 2A. (B) The effect of TTX on retinogeniculate projections. Pups were treated monocularly with TTX or saline from P10 to P14. CTB was injected into the other eye, and coronal sections of the dLGN ipsilateral to the CTB-injected eye were prepared at P15. The sizes of the CTB-positive areas relative to those of the dLGNs were measured. The data of control and ME-treated animals are the same as those in Figure 3. (**) P<0.01, (*) P<0.05, (N.S.) not significantly different (P≥0.05), unpaired Student's t-test or unpaired Welch's t-test (see Materials and Methods). Error bars represent S.D. The numbers in parentheses indicate the number of animals.