Degeneration in the DBA/2. (A) Cross-sections through proximal (Left) and distal (Right) optic nerve of a 13-mo DBA/2 shows degenerating profiles (arrowheads, bracket). (B) Degenerating profiles per section for three ages (n = 5–6). (C) Ratio of distal to proximal degenerating profiles decreases with age. (D) Rostral SC from 3-mo (Left) and 18-mo (Right) DBA/2 mice show comparable Black Gold II–stained RGC axon myelin in layer III despite opposing fractions of intact SC map (Insets). Lateral (L) and dorsal (D) are indicated. (E) Volume of superficial SC reconstructed from serial sections changes very little across ages and range of intact CTB mapping. (Inset) Average SC volume across age groups. (Scale bars: 10 μm in A, 200 μm in D.)

Transport loss in acute glaucoma with age. (A) IOP in young (3–4 mo) and aged (7–9 mo) rats is elevated acutely by 40% to 45% with microbead injection (circles) compared with saline solution in the opposite eye (diamonds). IOP is the same for young and aged rats for both the saline (P = 0.66) and microbead (P = 0.77) eye, whereas the microbead-induced elevation was significant for both age groups (P < 0.001). (B) Maps of CTB label for two young rats (Left) were intact, whereas two aged rats (Right) had deficits for the microbead-injected eye of 80% and 60%, respectively. Rostral (R) and medial (M) are indicated. (C) Fraction of intact SC retinotopic map (≥70% CTB signal density) was reduced in aged (P < 0.001, n = 5) but not young (P = 0.82, n = 5) microbead-injected rats. (Scale bar in B: 500 μm.)

Transport deficits in the DBA/2 SC. (A) Cross-sections through a 5-mo C57 brain showing normal RGC transport in the superficial SC (dotted lines) and in the nucleus of the optic tract (NOT) following unilateral injection of CTB (green). At far right is a complete retinotopic SC map with representation of optic disk gap (circle) and location of representative cross-sections (black dotted lines). Dorsal (D), rostral (R), caudal (C), and medial (M) are indicated. (B) SC of 5-mo DBA/2 with complete CTB label and corresponding retinotopic map. (C) SC of 8-mo DBA/2 with a 25% deficit in CTB middle to caudal (arrows) and resulting map. (D) SC from 10-mo DBA/2 with marginal retention of CTB label (arrowheads) shows an 88% retinotopic deficit. For DBA/2, IOP in mmHg is indicated for the corresponding eye. CTB signal density varies from 100% (red) to 50% (green) to 0% (blue). (Scale bars in D: 500 μm for sections or maps.)

Structural persistence after transport loss in DBA/2. (A) Three-month SC after bilateral CTB injection shows signal on one side with a deficit on the other (Upper). ERRβ labeling of RGC axon terminals in same section persists on both sides (Lower). (B) Ten-month left and right SC with severe loss of CTB signal (Upper) shows ERRβ beginning to dissipate (Lower), especially on side with no signal (arrowheads). (C) Seventeen-month left and right SC with ERRβ-labeled RGC axon terminals on one but not the other side (Upper). ERRβ-labeled RGC axons persist in optic tract (OT) for both (Lower). Arrows show direction of terminals. (D) Section of 8-mo SC with modest CTB signal deficit on right (arrowheads, Upper) shows full complement of RGC presynaptic terminals labeled for VGluT2. (E) Eighteen-month SC with complete bilateral loss of signal (Left). VGluT2-labeled RGC terminals persist on the left but not the right (dashed lines). (F) Fraction of SC volume with intact ERRβ label persists across ages even with severely depleted CTB signal maps (Left), as does the fraction containing intact VGluT2. (Scale bars: 500 μm in E and in SC sections; 50 μm in C and in OT.)

Sectorial transport deficits. (A) Map of CTB in 5- and 10-mo C57 SC with optic disk gap (circle) and rostral (R) and medial (M) are indicated. CTB maps in DBA/2 SC for 3-mo (B), 5-mo (C), 8-mo (D), and 10-mo (E) mice with deficits progressing from rostral or caudal edge to the optic disk gap (arrows). IOP in mmHg is indicated. (Scale bar: 500 μm.) (F) Fraction of SC retinotopic map with ≥70% CTB signal density versus age for individual SC (open circles). Five- and 10-mo C57 SC shown in a single group (n = 3 each). Individual SC separated horizontally for clarity. Mean ± SE is shown (diamonds). DBA/2 transport differs from C57 at 8 mo (P = 0.05, n = 10), 10 mo (P = 0.004, n = 13), 12 mo (P < 0.001, n = 10), and ≥15 mo (P < 0.001), but not for 3 mo (P = 0.13, n = 11) or 5 mo (P = 0.16, n = 12). For ≥15 mo, n = 16 (all 0 signal), but only 10 points shown for clarity.

Distal to proximal transport loss. (A) Ten-month SC section and complete retinotopic map with optic disk gap (circle). Ventral/dorsal LGN and OPT anterior to the SC with intact CTB. Lateral (L), medial (M), rostral (R), and dorsal (D) are indicated. (B) CTB absent in 10-mo SC but retained in LGN and OPT. (C) CTB present in dLGN and OPT but not vLGN of 10-mo brain with no SC signal. (D) A 12-mo brain with no signal in SC and LGN but some in OPT. (E) Optic tract and nerve from 10-mo brain with no signal shows CTB+ axonal dystrophies. Direction of axon terminals indicated (distal). (F) In 12-mo animal with no brain signal, CTB collects in axonal dystrophies (arrowheads) of optic nerve labeled for myelin basic protein (MBP) after penetrating beyond the unmyelinated nerve head. Bracketed region at higher magnification (Right) with dystrophic axons (arrowheads) extending toward the brain (arrow). (Scale bars: 500 μm in B and SC cross-section in A; 250 μm in D and LGN and OPT in A–D; 20 μm in E; and 200 μm in F.)

Active uptake persists. (A) Twelve-month retina has some CTB+ axons (red) near optic nerve head (ONH). Corresponding SC contained no CTB signal. (B and C) Two locations from A labeled for α-tubulin (Tub). Region in B has both intact CTB transport (bracket) and uptake (arrowhead); region in C has only uptake (arrowheads). (D) Ten-month retina with residual CTB (red); SC contained none. (E and F) Two locations from D labeled for phosphorylated heavy-chain neurofilament (SMI-31). Region in E has both intact transport (brackets) and uptake (arrowhead); region in F shows a single transporting axon (bracket). (G) Five-month retina shows extensive CTB uptake in labeled RGCs. CTB is compartmentalized in the endoplasmic reticulum before transport and thus does not colocalize strictly with SMI-31 (Inset) (21). Green signal has been adjusted down to decrease background. (H) Brn3a-labeled RGCs (red) in a 12-mo retina contain CTB (green; arrowheads). Blue levels (SMI-31) adjusted to accent nontransporting axons. Brain contained no CTB. (I) RGCs with CTB uptake (%) versus fraction of intact SC map. Cells quantified in fields from the two hemiretinas (gray bars) in eight eyes (mean ± SD). Age (mo) and level of uptake required to match SC transport (dotted lines) are indicated. Asterisk marks only hemifield with uptake below transport. (Scale bars: 200 μm in A and D; 20 μm in B, C, and E–G; 5 μm in H.)