Cytochrome oxidase and neurofilament reactivity in monocularly deprived human primary visual cortex

Abstract

Previous studies of human primary visual cortex (V1) have demonstrated a significant eye-specific decrease in cytochrome oxidase (CO) staining following monocular enucleation. We have extended these results by examining CO staining and neurofilament labeling in V1 from a patient with long-standing monocular blindness. A pattern of reduced neurofilament reactivity was found to align with pale CO-stained ocular dominance columns. Neurons located within deprived ocular dominance columns were significantly smaller compared with those in nondeprived columns. A spatial analysis of the relationship between CO blobs and ocular dominance columns revealed that both deprived and nondeprived blobs tended to align with the centers of ocular dominance columns.

Figure 1

Images of the optic nerves (top: Luxol fast blue/H&E stain for myelin) and lateral geniculate nucleus (bottom: Nissl stain) provided support for the clinical report that the lesion was monocular (right eye) and long standing. The deprived-eye's optic nerve (A) was weakly stained and showed a pronounced loss of myelin compared with the contralateral optic nerve (B). A similar result was observed in sections from the left lateral geniculate nucleus where deprived layers were weakly stained (C) and, at high magnification, exhibited evidence of neuronal atrophy, with preservation of neurons in nondeprived layers (D). Scale bars = 250 μm (A, B), 500 μm (C), 100 μm (D).

Figure 2

Low-magnification (top) and high-magnification (bottom) photomicrographs of nonadjacent coronal V1 sections through the posterior aspect of the cuneus revealed a distinct laminar pattern when stained for CO (A, C) or labeled for nonphosphorylated neurofilament (B, D). CO staining was found in all layers but was heaviest within layer IVC. Neurofilament labeling was strong in layers II/III, IVB, V, and VI, whereas layer IVC was weakly labeled. For both markers, alternating patches of dark and light reactivity were found within each layer of V1 and this pattern was aligned across layers. High-magnification photomicrographs (C, D) are from the regions in (A, B) that are highlighted with arrows. The asterisks in (A, B) identify the approximate V1/V2 boundary. This border was determined using area-specific anatomical characteristics that have been described in neurofilament-labeled sections from the human visual cortex (Hof and Morrison 1990). Scale bars = 1 mm.

Figure 3

Sections cut tangential to the cortical surface revealed alternating dark and light regions of CO staining in layer IVC (A) and neurofilament labeling in layer IVB (B). These sections were aligned using the pattern of radial blood vessels, and the reaction patterns were compared by calculating optical profiles through the same regions in each section (along a line joining the 2 asterisks in A and B). The profiles for CO (black dotted line) and neurofilament (gray dotted line) showed substantial overlap (r = 0.77). Thus, the same regions of V1 have reduced CO staining and neurofilament labeling. Scale bar = 1 mm.

Figure 4

Adjacent sections cut tangentially and labeled for neurofilament (A)or stained for Nissl substance (B) were aligned for comparison using the pattern of radial blood vessels. The neurofilament section contained distinctly labeled multipolar cells that identified it as having been cut through layer IVB. Neurofilament labeling revealed a distinct pattern of ocular dominance that consisted of alternating regions of dark (nondeprived) and light (deprived) immunoreactivity. The adjacent section assessed for Nissl substance showed homogeneous staining at low magnification and did not exhibit an ocular dominance pattern. Arrows in this figure point to overlapping deprived (hollow arrows) and nondeprived (filled arrows) areas for the 2 markers. Scale bar = 1 mm.

Figure 5

High-magnification photomicrographs of the regions highlighted with arrows in Figure 4. Significantly, fewer neurofilament-labeled somata and dendrites were found within parts of V1 serving the deprived eye (A) when compared with parts serving the nondeprived eye (B). No obvious difference in staining intensity between deprived and nondeprived areas was observed after Nissl staining (C, D). Quantification of staining intensity (E) and numeric density (F) showed no statistical difference between deprived and nondeprived regions (P = 0.3 and P = 0.6, respectively); however, the cross-sectional area of neuron somata (G) within deprived parts of V1 was significantly smaller than somata within nondeprived parts (P = 0.02). Arrows in this figure point to overlapping blood vessels between sections. Double asterisks indicate significant difference. Error bars represent SEM. Scale bar = 100 μm.

Figure 6

This figure shows a series of adjacent sections at different depths through V1 that were reacted for either CO (left) or neurofilament (right). Sections through layer II/III (A) and IVB (C) that were reacted for CO showed regularly spaced blobs of reactivity. A bridge of pale reactivity connected darkly stained CO blobs within nondeprived ocular dominance columns, whereas blobs in deprived columns were often isolated and stained less intensely. Neurofilament labeling within layers II/III (B) and layer IVB (D) was organized into alternating dark and light regions. Layer IVC showed strong reactivity for CO but was weakly labeled for neurofilament (E, F). CO staining within layer IVC (E) was organized into distinct dark and light areas that aligned with the pattern of CO staining and neurofilament labeling in the more superficial layers. Neurofilament labeling in layer IVC was weak and revealed only a faint ocular dominance pattern (F). Sections through layer VI were lightly stained for CO and showed a weak ocular dominance pattern (G). Neurofilament labeling in layer VI was heavy and showed a low-contrast ocular dominance pattern (H). Scale bar = 2 mm.

Figure 7

The spatial relationship between CO blobs and ocular dominance columns is demonstrated in this figure. A section through the superficial layers that contained CO blobs (A) was aligned with a section through layer IVC that contained a pattern of ocular dominance columns (B). Blobs were identified using an automated computer program that calculated relative optical densities throughout the section (C) and then plotted filled contours representing maximum-to-minimum staining intensities across the section. Markers (asterisks) representing computer-identified blob centers (max staining) were aligned with the map of ocular dominance, and then blob position within the map was evaluated. The gray asterisks in (D) represent blobs that could not be related to ocular dominance columns because surrounding column borders were not clear. Scale bar = 1 mm.

Figure 8

This figure shows an example of the spatial relationship between blobs within the superficial layers (A) and ocular dominance columns in layer IVC (B) taken from a different region of V1. CO blobs were identified using a computer program that plotted filled contours representing maximum-to-minimum staining intensities (C), and an asterisk was positioned at the center of each computer-identified blob (D). The asterisks representing blobs were spatially aligned with the ocular dominance map to examine the relationship between these 2 features. The gray asterisks in (D) represent blobs that could not be evaluated because of unclear ocular dominance borders. Scale bar = 1 mm.

Figure 9

Quantification of the spatial relationship between CO blobs and ocular dominance columns. These graphs plot the distribution of nondeprived (A) and deprived (B) blob position within an ocular dominance column, from the center of a column to the border. The distance from the center of a blob to the nearest column border was calculated and divided by half of the total column width at that position. This provided a number that ranged from 1, for blobs aligned with the center of an ocular dominance column, to 0, for blobs aligned with a border. The distribution of positional measurements for both nondeprived and deprived blobs was skewed to the left, indicating that the majority of blobs were located near the center of an ocular dominance column and few were positioned along a border. A chi-square statistic revealed that the distribution of deprived and nondeprived blobs was significantly different from that expected by a random distribution (P < 0.0001). The dashed line represents the blob percentage for each column position that would be expected if blobs were randomly distributed across the ocular dominance map. Error bars represent standard deviation.