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Necessity for Afferent Activity to Maintain Eye-Specific Segregation in Ferret Lateral Geniculate Nucleus

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Science  31 Mar 2000:
Vol. 287, Issue 5462, pp. 2479-2482
DOI: 10.1126/science.287.5462.2479

Abstract

In the adult mammal, retinal ganglion cell axon arbors are restricted to eye-specific layers in the lateral geniculate nucleus. Blocking neuronal activity early in development prevents this segregation from occurring. To test whether activity is also required to maintain eye-specific segregation, ganglion cell activity was blocked after segregation was established. This caused desegregation, so that both eyes' axons became concentrated in lamina A, normally occupied only by contralateral afferents. These results show that an activity-dependent process is necessary for maintaining eye-specific segregation and suggest that activity-independent cues may favor lamina A as the target for arborization of afferents from both eyes.

Development of mammalian visual pathways is characterized by activity-dependent sculpting of precise neuronal connections from initially diffuse projections. In the developing lateral geniculate nucleus (LGN), retinal ganglion cell inputs from the two eyes initially overlap and only later segregate into eye-specific layers (1,2). This eye-specific segregation in the LGN is activity dependent; it is prevented by blocking action potentials in the LGN (3) or by binocular blockade of spontaneous retinal activity (4, 5).

The failure to develop eye-specific segregation in the absence of activity appears to be due to a specific role for neuronal activity in a competitive process. Activity is not necessary for the general growth and development of axons. Activity blockade does not freeze individual axons in an immature, sparsely branched state. Instead, in the absence of activity, retinogeniculate axons continue to grow and elaborate their axonal arbors, albeit without spatial specificity (3). Monocular deprivation paradigms have suggested that the activity-dependent processes that cause segregation of eye-specific layers in the LGN involve competition between afferents serving the two eyes. If the normal balance of activity between the two eyes is disrupted pharmacologically by monocularly silencing either retinal waves (4) or retinal ganglion cell action potentials (5), the silenced eye loses territory in the LGN, while the normal eye's projection expands.

Previous studies have concentrated on the initial establishment of segregation. If binocular activity blockade is begun early in development when the afferents serving the two eyes still have overlapping projection patterns, eye-specific axonal segregation is prevented or retarded (3–5). To investigate whether activity-dependent competition is necessary not only for establishment of eye-specific segregation but also for its maintenance, we blocked binocular retinal activity beginning after the initial formation of eye-specific layers in the LGN.

In the ferret LGN, eye-specific segregation of retinal axons is complete by postnatal day (PND) 9 (2) (Fig. 1A). Further segregation of afferents into ON- and OFF-center sublaminae is complete by PND 25 (2) (Fig. 1B). Binocular activity from PND 9 to PND 25 was blocked in six ferrets by daily binocular intravitreal injections of a high concentration of the glutamate analog 2-amino-4-phosphonobutyric acid (APB) (6). To ensure full blockade of both ON- and OFF-center retinal ganglion cell activity, a retinal concentration of 3.5 mM APB was used. This concentration was found to block all activity in the LGN of even relatively mature (PND 30) ferrets for 24 hours or more (Fig. 2, A and B) and much lower doses of APB block spontaneous activity in the retina of young ferrets (7) (Fig. 2C). The chronic APB treatment did not appear to produce any morphological damage to the developing retina (8) (Fig. 2D). Daily eye injections of saline or of relatively low dosages of APB (700 μM) (9) or systemic injections of APB did not affect development of the retinogeniculate afferent projection (Fig. 2E).

Figure 1

Normal development of the LGN. Retinogeniculate afferents from the right eye were labeled by anterograde transport of WGA-HRP. (A) Horizontal sections from PND 9 ferret LGN show good segregation of afferents from the two eyes into lamina A (contralateral) and lamina A1 (ipsilateral). (B) PND 25 ferret afferents show both eye-specific segregation into lamina A and lamina A1 and ON/OFF segregation into inner (Ai and A1i) and outer (Aoand A1o) leaflets. C laminae are also seen. Bar = 100 μm.

Figure 2

Physiological effects of APB treatment and controls for nonspecific effects of eye injections and possible systemic APB effects. (A) APB (3.5 mM) abolishes both ON and OFF LGN responses. Poststimulus time histograms of multiunit LGN activity in response to flashing light and dark circles (white, 1.5 s; black, 1.5 s). (Top left) Normal PND 30 multiunit ON response from lamina A. (Bottom left) Recording from the same location 20 min after contralateral eye APB injection. (Top right) Normal PND 30 multiunit OFF response from lamina A1. (Bottom right) Recording from the same location 20 min after ipsilateral eye APB injection. yaxis, 700 spikes per second; x axis, 3 s. APB was found to block all ON and OFF activity in the PND 30 LGN for 24 to 28 hours in three ferrets. (B) APB (3.5 mM) blocks spontaneous LGN activity in PND 30 ferrets. Ten-second raster plots of spontaneous LGN activity. (Top) Normal spontaneous activity in layer A outer. (Middle) Activity at same location 10 min after contralateral eye APB injection. (Bottom) Thirty min after injection. (C) Effects of 1 μM APB on spontaneous retinal activity in PND 7 ferret. In vitro calcium imaging shows complete blockade of retinal ganglion cell activity. y axis, nM Ca2+; x axis, 40 min. APB was applied at 19 min and washed out at 29 min. Data graciously provided by Wong and Wong (16). (D) APB does not disrupt normal development of gross morphology in the retina. Thionin-stained sections from ferret retina treated with 3.5 mM APB from PND 9 to 25 (right) and from a normal PND 25 animal (left). Bar = 50 μm. (E) Eye injections of saline or low concentration APB or systemic injections of high-concentration APB do not affect retinogeniculate afferent segregation. Binocular WGA-HRP injections reveal the normal PND 25 pattern of eye-specific layers and ON/OFF sublaminae in ferret LGN. Daily eye injections of 0.9% NaCl or 700 μM APB PND 9 to 25 do not alter the projection pattern. Note that 700 μM APB is sufficient to block ON-center cells in PND 30 ferrets for approximately 24 hours, suggesting that OFF-center activity alone may be sufficient to drive axonal segregation in the LGN. Systemic daily (PND 9 to 25) injections of APB directly into the cisterna magna in three ferrets had no effect. The concentration of APB used was the same as that used for eye injections, but the volume was doubled so the total amount of APB administered in cisternal injections was equal to that administered in binocular eye injections. Bar = 100 μm.

After the APB-induced binocular activity blockade from PND 9 to 25, the pattern of retinal ganglion cell arborization in the LGN (10) was very different from that seen in normal PND 25 animals (compare Fig. 3A and Fig. 1B). Instead of the normal pattern of eye-specific segregation into lamina A (contralateral) and lamina A1 (ipsilateral), the LGNs of APB-treated ferrets show spatially identical contralateral and ipsilateral projection patterns, indicating complete overlap of the axons from the two eyes (Fig. 3). This intermingling of inputs from the two eyes cannot represent the preservation of an immature, unsegregated projection pattern, because the afferents were already segregated into eye-specific layers at PND 9 (Fig. 1A). Instead, activity blockade caused a desegregation of afferents that were previously segregated.

Figure 3

Effects of APB- induced retinal activity blockade on eye-specific segregation of afferents in the LGN. Retinogeniculate afferents were labeled by anterograde transport of WGA-HRP. (A) Horizontal sections from the LGN contralateral (left) and ipsilateral (right) to the injected eye show the same pattern of afferent projection. Cross-sectional areas of the ipsilateral and contralateral projections to lamina A measured in 10 horizontal sections through the middle of LGNs from four APB-treated animals were statistically indistinguishable (two-tailedt test; P < 0.001) from each other and from the area occupied by the contralateral projection to lamina A in similar sections from four normal ferrets [APB contralateral, 0.56 ± 0.06 mm2; APB ipsilateral, 0.49 ± 0.08 mm2; normal contralateral, 0.52 ± 0.07; normal ipsilateral (lamina A1), 0.18 ± 0.04]. Faint segregation of afferents into inner (Ai) and outer (Ao) leaflets can be seen in both eyes' projections. Staining was always darker contralateral to the injected eye, suggesting that the contralateral projection remains stronger than the ipsilateral projection. (B) Sections from the ventral third of the LGN of another ferret again show identical projection patterns contralateral (left) and ipsilateral (right) to the injected eye. This abnormal lack of segregation was found in all six animals studied throughout the depth of the LGN. Bar = 100 μm.

APB-treated LGNs showed a lack of the interlaminar space (11) (Fig. 4) similar to that seen when animals were binocularly (12) or monocularly (13) enucleated before development of LGN lamination. In the present experiments, however, a faint interlaminar space is already present at PND 9 (2) (Fig. 4A) when the APB treatment began, indicating that maintenance of the interlaminar space, like that of axon segregation, is activity dependent.

Figure 4

APB-induced retinal activity blockade disrupts development of the interlaminar space in ferret LGN. (A) Faint cell-sparse space between lamina A and lamina A1 can be seen in normal PND 9 ferret LGN (arrows). (B) By PND 25, the interlaminar space is clear. (C) APB-treated ferrets show no interlaminar space on PND25. Nissl-stained section with arrows marking the apparent A/A1 border, as shown by the pattern of the afferent projection seen in an adjacent WGA-HRP section from the same LGN (D). Bar = 100 μm.

Despite the lack of eye-specific segregation of afferents after PND 9 to 25 activity blockade, the afferent projections were stereotyped in their patterning and did not represent a random diffuse projection to the entire LGN as when activity is blocked before development of segregation. Instead, the projections from both eyes were concentrated in a region similar to the normal location of lamina A (Fig. 4, C and D) and appeared to avoid lamina A1, indicating a substantial expansion and relocation of the afferent projection from the ipsilateral eye. This spatially restricted but unsegregated pattern suggests that there might be activity-independent cues in lamina A that are relatively attractive to axons from both eyes. In normal animals, activity-dependent competition occurs, and contralateral axons appear to have a competitive advantage, allowing them to take over the attractive real estate of lamina A and force the ipsilateral axons into lamina A1. Without competition, both eyes' axons would have equal ability to arborize in lamina A and select this region preferentially over lamina A1. The preferential arborization of axons in lamina A seen in this study was not observed in previous studies in which the development of segregation was prevented or retarded by activity blockade or enucleation before establishment of eye-specific layers (3–5, 12, 13). This difference could be explained if a preference for lamina A is established during the initial axonal segregation (PND 0–9) and requires a period of normal neuronal activity.

This study indicates that activity-dependent competition is vital not only for initial establishment of specific connections in the mammalian visual system but also for maintenance of these connections at least for some time during development. The possibility of attractive molecular cues or gradients in lamina A and the interactions between such cues and activity-dependent competition in normal development remain important open questions.

  • * To whom correspondence should be addressed. E-mail: bxchapman{at}ucdavis.edu

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