Stereotyped lamina-specific axonal refinement in the SC
To study the cellular mechanisms that underlie lamina-specific axonal refinement, we used CB2-GFP transgenic mice. In this mouse line, GFP is selectively expressed by OFF-αRGCs. This allows visualization of OFF-αRGC axons in retinorecipient targets across development and into adulthood (Huberman et al., 2008a
). Previous studies have shown that from postnatal day 0 (P0) until P8 RGC axons undergo retinotopic refinement, with the major phase of anatomical remodeling occuring between P0–P4 (McLaughlin and O’Leary, 2005
). Also from P0–P4, SC neurons complete their migration into the appropriate laminae (Edwards et al., 1986
) (). Subsequently, lamina-specific refinement ensues: from P4–P6, OFF-αRGCs project axons broadly across the depth of retinorecipient SC and then from P7–P12, OFF-αRGC axons are removed from the upper layer of the retinorecipient SC (uSGS). Thus by P12, virtually all OFFαRGC axons are found in the lower SC retinorecipient layer (lSGS) () (Huberman et al., 2008a
). The stereotyped refinement of axons arising from a RGC population expressing a stable genetic marker provides a unique opportunity to examine the ultrastructural changes associated with lamina-specific axon targeting and to directly compare the ultrastructure of RGC axon projections destined for removal versus those destined for laminar stabilization.
Ultrastructure of lamina-specific projections to the SC during refinement
To explore the ultrastructural changes that underlie lamina-specific axonal refinement, we analyzed GFP expressing (GFP+) OFF-αRGC axons in the SC of developing mice, using serial-section immuno-EM. First we focused on axonal boutons because they are a well-defined structural correlate of presynaptic terminals. GFP+ axons in the superficial-most 150um of the SC are defined as axons in the uSGS that are fated for removal, because virtually all such profiles are pruned from this lamina by P11/12 () (Huberman et al., 2008a
). EM analysis revealed that GFP+ boutons in the uSGS increased in average size by 83% between P4 and P6, followed by a continuous decrease in average size (~41%) from P6 to P11 (; P4/P6 P
=0.003, P6/P11 P
=0.007, one-way ANOVA Hochberg tests; n=12–17). By contrast, the average size of GFP+ boutons in the lSGS (those fated for stabilization) remained stable throughout the major phase of lamina-specific targeting (; P
=0.20, one-way ANOVA; n=12–18). Interestingly, throughout the period of laminar refinement (P4–P11), GFP+ boutons in the uSGS were significantly smaller than the GFP+ boutons targeted to the lSGS (; P
=0.01, two-way ANOVA; n=56–62). This could arise because the more distal portions of the GFP+ axons are thinner, due to molecular differences in boutons fated for removal versus stabilization (see discussion), or both. Regardless, our data indicate that axon boutons that are transiently targeted to inappropriate layers fail to achieve the full size of axon boutons that are targeted to their correct lamina.
Ultrastructure of boutons formed by developing OFF-αRGC axons in the SC
Structural synapse formation and elimination underlie lamina-specific targeting
Next we asked whether lamina-specific refinement involves synapse formation and elimination. We defined a synapse as a GFP-labeled profile with: i) a GFP+ bouton containing synaptic vesicles, ii) an opposing synaptic cleft and iii) a postsynaptic density. Using those criteria, we first observed GFP+ synapses in the retinorecipient SC at P6. At this age, synapses were present in both uSGS and lSGS (). Indeed, the percentage of GFP+ boutons making synapses was very similar in the uSGS versus lSGS at P6 (; uSGS, 47% (n=7/15); lSGS, 50% (n=8/16)). From P8 to P11, most GFP+ axons were removed from the uSGS, but a minority persisted there (). Interestingly, the GFP+ axons that persisted in the uSGS contained synapses at percentages only slightly lower than GFP+ inputs to the lSGS at the same age (P8: uSGS 47% (n=8/17), lSGS 56% (n=9/16); P11: uSGS 42% (n=5/12), lSGS 50% (n=9/18)). Overall, these data indicate that early in development, OFF-αRGC axons form synaptic contacts with cells in both correct and incorrect target laminae. Given that virtually all GFP+ axonal inputs to the uSGS are removed by P12 (), lamina-specific refinement of OFF-αRGC axons must involve synapse elimination.
Axonal degeneration during lesion-induced but not developmental lamina-specific pruning
How are OFF-αRGC axons and synapses removed from the uSGS? Neither our fluorescent microscopic nor EM analysis revealed any immediate signs of axonal degeneration or glial engulfment during laminar retino-SC refinement ( and ). In CB2 mice, GFP+ OFF- αRGCs in each eye project only to the contralateral SC, so to more closely evaluate whether lamina-specific refinement involves axon degeneration, we removed one eye from P8 CB2-GFP mice and compared the GFP+ axons in the SC ipsilateral and contralateral to the enucleated eye. Four hours post-enucleation, fragmented GFP+ axons were readily observed in the contralateral (deafferented) SC but not in the ipsilateral (control) SC (). The axon fragmentation was also accompanied by a significant increase in Iba1+ microglia 4–6 hours post-enucleation (; P
=0.03, two-way ANOVA; n=3 for each time point). Moreover, in CB2-GFP mice that also lacked the immune protein C1q (CB2-GFP::C1q−/−
), lamina-specific pruning proceeded normally (). C1q is important for microglia-mediated synapse elimination during eye-specific refinement (Stevens et al., 2007
). Thus, that the lack of impairment in lamina-specific pruning in CB2-GFP::C1q−/−
mice further supports the idea that this process is glia-independent. Indeed, when we compared the ultrastructure of GFP+ boutons in the uSGS 5 hours post-enucleation, intact boutons with synapses were observed in the ipsilateral (non-deafferented) hemisphere of the SC (). By contrast, contralateral to the enucleated eye, degenerating GFP+ fragments surrounded by electron-lucent glial cell processes, were readily apparent (; and see Supplemental movie
). Collectively, these data indicate that during normal development, laminar specificity is achieved independently of axonal degeneration or glial engulfment.
Decoupling synaptic maintenance and axonal refinement
Our findings that synapse formation and elimination are concurrent with the refinement of OFF-αRGC axons (–) led us to ask whether the formation and/or maintenance of synaptic contacts is related to the stability of RGC axonal arbors. We reasoned that if synapse formation is a prerequisite for stabilizing a given arbor, then synaptic maturation might progress faster for boutons in the lSGS versus the uSGS. Interestingly, in both the uSGS and lSGS, when the synaptic profiles were present, on average 1 to 2 synaptic contacts were associated per bouton (uSGS: P6 1.00±0.00, P8 1.38±0.18, P11 1.40±0.16; lSGS: P6 1.25±0.12, P8 1.11±0.08, P11 1.22±0.10; n=5–9). Moreover, PSD lengths were comparable for GFP+ synapses regardless of whether they resided in the uSGS or lSGS (; P=0.33, two-way ANOVA; n=5–9). Indeed, there was no significant increase in synaptic number or size for deep layer boutons during the major phase of arbor stabilization (synaptic number: P=0.76, one-way ANOVA; synaptic size: P=0.12, one-way ANOVA; n=8–9). These results indicate that the structural maturation of synapses fated for removal highly resembles the maturation of synapses fated for stabilization.
Quantification of OFF-αRGC axonal boutons in the developing SC
Our observation that bouton size reduced from P6–P11 () supports the notion that axon retraction is the cellular mechanism for lamina-specific refinement. If the balance of axon growth and retraction is determined primarily by the presence of synapse on a given arbor, then the absence of a synapse might correlate with, and perhaps even predict, a reduction in bouton size. Although we did not analyze individual boutons repeatedly over time using time-lapse imaging, comparison of GFP+ boutons from different aged mice revealed that bouton size in the lSGS did not vary significantly as a function of synaptic contact (; P=0.72, two-way ANOVA, n=5–9). In the uSGS, GFP+ boutons steadily decreased in size from P6 through P11, regardless of whether they contained a synapse or not (; age P=0.02, synapse P=0.36, two-way ANOVA; n=5–9). These data indicate that synaptic parameters are not correlated with the stability of OFF-αRGC axonal arbors during lamina-specific refinement.