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First published online 23 June 2004
doi: 10.1242/dev.01212

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ERK signaling is required for eye-specific retino-geniculate segregation

Sibel Naska^1,*,, Maria Cristina Cenni², Elisabetta Menna² and Lamberto Maffei^1,2

¹ Scuola Normale Superiore, piazza dei Cavalieri 7, 56100 Pisa, Italy
² Istituto di Neuroscienze del CNR, via G. Moruzzi 1, 56100 Pisa, Italy

View larger version (68K): [in a new window]   Fig. 1. Development of retino-geniculate connectivity. Representative coronal sections through dLGN of P4 (A-D, n=3) and P9 (E-H, n=3) rats. Fibers from the ipsilateral projecting eye are labeled with CTB-Alexa Fluor 488 (green), whereas the projections from contralateral eye are labeled with CTB-Alexa Fluor 594 (red). (I,J,K) Quantification of relative contralateral (I), ipsilateral (J) and overlap (K) areas of P4 (n=3) and P9 (n=3) rats. The areas occupied by ipsilateral and contralateral projections are significantly different between P4 and P9 groups (Student's t-test, P<0.05 for contralateral and ipsilateral areas). Note that the overlap area decreases from 20% in P4 to 2% in P9 dLGN (K). At P4, high magnification of the area of overlap shows that fibers from both eyes are largely intermixed (D), whereas by P9, high magnification of the border between the two projections shows that these fibers terminate in distinct regions (H). (L-O) pERK expression in the dLGN is developmentally regulated. pERK immunohistochemistry in the dLGN at different postnatal stages reveals an increasing expression of pERK from P0 (L, n=3) to P5 (M, n=4). Note the reduced staining at P8 (N, n=3) and P11 (O, n=3). pERK-positive cells (green) co-express the neuronal marker NeuN (red), (P5 rats, n=4, P). Error bars depict the s.d. Scale bars: 200 µm for A-C,E-G; 10 µm for D,H; 100 µm for L-O; 50 µm for the insets; 25 µm for P.

View larger version (64K): [in a new window]   Fig. 2. Effects of the inhibitory action of U0126 and PD98059 in retino-geniculate connections. Intracerebroventricular (ICV) injection of U0126 (500 µM, P5 rats n=4, B) or PD98059 (1 mM, n=4, C) drastically reduces ERK activation in the dLGN with respect to the saline injection (n=3, A). (D-G) U0126 does not affect pCaMKII or pAkt expression. ICV injection of saline (D) or 1 µl of 500 µM U0126 (E) does not block CaMKII phosphorylation in the dLGN. Also pAkt levels in the dLGN remain unchanged after ICV injection of saline (F) or U0126 (G, P4 rats, n=5 for each treatment). (H-M) Distribution of retinal projections in the dLGN of P9 rats that received beads-U0126 or beads-vehicle ICV injections. (H,H') Projections from the contralateral eye expand filling the entire dLGN. (I) The relative contralateral area of U0126-treated animals (n=6) is significantly larger than that of control animals (n=5; Student's t-test, P<0.05). (J,K) Ipsilateral projections expand occupying an area twice greater than that of control animals (Student's t-test, P<0.05). (L,M) The projections coming from the two eyes largely overlap in the U0126-treated animals. Error bars depict the s.d. Dashed lines indicate the relative eye-specific areas in normal P9 rats. Scale bars: 100 µm for A-G; 200 µm for H,J,L; 100 µm for H'.

View larger version (59K): [in a new window]   Fig. 3. Specific blockade of pERK in LGN neurons. (A) All pERK-positive cells (red labeling) in the dLGN of P5 rats (n=5) co-localize with dLGN projection neurons labeled with the retrograde tracer FluoroGold (green labeling). (B) Schematic representation of visual cortex injections of fluorescent latex beads in newborn rats. (C) A large number of retrogradely labeled neurons are evident in the dLGN after injections of red fluorescent latex beads in the primary visual cortex (n=4). (D) The red beads are present throughout the cytoplasm of geniculo-cortical neurons labeled by Fluorogold (n=3). (E) Cortical injections of beads-U0126 produce a prolonged blockade of ERK activation in the dLGN ipsilateral to the injected side. Forty-eight hours after injection, no detectable pERK staining (green) is observed in LGN neurons with red beads inside the cytoplasm (E, n=7). (F) Unilateral cortical injections of beads-U0126 do not affect pERK staining in the dLGN contralateral to the injected cortex; indeed, no red beads are observed in this side of the brain (n=7). (G,H) Injections of beads-U0126 in the left side of visual cortex do not affect pERK expression in the eye. Coronal sections from the retina of the contralateral eye, which sends the major afferents to the LGN, are shown. pERK immunostaining in the RGCs of treated animals (G, n=7) is not different from that of controls (H, n=3). Scale bars: 10 µm for A,E,F; 100 µm for C; 25 µm for D; 20 µm for G,H.

View larger version (40K): [in a new window]   Fig. 4. dLGN projection neurons contribute to the development of retino-geniculate connectivity. (A-E) Coronal sections of dLGN in P9 rats (n=18) that received injections of retrogradely transported beads-U0126 in the primary visual cortex, and quantification of relative contralateral, ipsilateral and overlap areas in the dLGN. Both ipsilateral (A) and contralateral (B) projections are diffusely distributed in the dLGN. The relative ipsilateral (C) and contralateral (D) areas of U0126-treated animals (n=18) are statistically greater than those of control animals (n=10), (Student's t-test, P<0.05 respectively). (E) The relative overlap area of U0126-treated animals is larger than that of control animals. Error bars depict the s.d. Dashed lines indicate the relative eye-specific areas in normal P9 rats. Scale bar: 200 µm.

View larger version (79K): [in a new window]   Fig. 5. Effects of binocular retinal injections of U0126. pERK staining in P5 rats is evident in all cells of the GCL injected with saline (A, n=3) but is strongly decreased after 60 minutes of U0126 treatment (B, n=4). (C) Control retinas with no primary antibody show background fluorescence. (D) Western blot analysis reveals that, at 48 hours after intravitreal injections of beads-U0126 (1 mM), pERK levels in the retina are decreased by 52±19% with respect to controls injected with beads-vehicle, Student's t-test, P<0.05 (C, control; T, U0126-treated). Each column is an average of six different samples. Each sample pools together two retinas. (E,F) In P5 rats, no differences in pERK levels within the dLGN are observed 48 hours after binocular injections of beads-vehicle (E, n=6) or beads-U0126 (F, n=6). (G-L) Eye-specific segregation in P9 rats that received binocular intravitreal injections of beads-U0126. (G,H) Both contralateral and ipsilateral projections are diffusely distributed in the dLGN. (J,K) The relative contralateral and ipsilateral areas of U0126-treated animals (n=7) are significantly larger than those of control animals (n=6), (Student's t-test, P<0.05, respectively). (I,L) The relative overlap area of U0126-treated animals is larger than that of control animals. Error bars depict the s.d. Dashed lines indicate the relative eye-specific areas in normal P9 rats. Scale bars: 25 µm for A,B,C; 50 µm for E,F; 200 µm for G,H,I.

View larger version (97K): [in a new window]   Fig. 6. Monocular retinal blockade of ERK activation. (A,B,D,E) Representative images of both sides of the dLGN of rats that received monocular injections of beads-U0126 (A,B; n=7) or vehicle (D,E; n=7). Projections from the treated eye are labeled with CTB-Alexa Fluor 488 (green) and those from untreated eye with CTB-Alexa Fluor 594 (red). Note that in the U0126-treated animals both contralateral (A) and ipsilateral (B) fibers from the treated eye (green) occupy smaller territories then the corresponding projections from the untreated eye (red, A,B). Conversely, in control rats (D,E), fibers from both eyes occupy similar areas in the dLGN. (C,F) Cresyl violet staining of cells in the GCL. Living cell density in the retinas of rats monocularly injected with U0126 (C; 69.3±6.1 cells per field, n=5) is not statistically different (Student's t-test, P>0.05) from that of vehicle-treated retinas (F; 66.1±2.5 cells per field, n=4). Scale bars: 200 µm for A,B,D,E; 20 µm for C,F.

View larger version (47K): [in a new window]   Fig. 7. Effects of concurrent pERK blockade in both retinas and geniculate neurons. In treated animals (n=10) the contralateral fibers fill nearly the entire nucleus (A,C), whereas those coming from the ipsilateral eye are largely diffused in the dLGN (B,D), overlapping through 18.5% of the total area (E). This distribution of fibers is significantly different from that of control animals (n=6; Student's t-test, P<0.05 for contralateral and ipsilateral). Error bars depict the s.d. Dashed lines indicate the relative eye-specific areas in normal P9 rats. Scale bar: 200 µm.

View larger version (98K): [in a new window]   Fig. 8. Effects of binocular retinal injections of epibatidine. (A,B) Representative coronal sections of dLGN in P9 rats that received binocular injections of epibatidine (n=3). The retinal fibers of only one eye labeled with CTB-Alexa Fluor 594 (red) are shown, as the other eye projection is identical. The contralateral fibers fill nearly the entire nucleus (A) and the ipsilateral projections are expanded considerably (B). (C,D,E,F) Forty-eight hours after binocular injection of epibatidine, the expression of pERK in retina and dLGN of P7 rats (C,E, n=4) is strongly decreased compared with controls injected with vehicle (D,F, n=3).

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