Aging Alters Daily and Regional Calretinin Neuronal Expression in the Rat Non-image Forming Visual Thalamus

Abstract

Aging affects the overall physiology, including the image-forming and non-image forming visual systems. Among the components of the latter, the thalamic retinorecipient inter-geniculate leaflet (IGL) and ventral lateral geniculate (vLGN) nucleus conveys light information to subcortical regions, adjusting visuomotor, and circadian functions. It is noteworthy that several visual related cells, such as neuronal subpopulations in the IGL and vLGN are neurochemically characterized by the presence of calcium binding proteins. Calretinin (CR), a representative of such proteins, denotes region-specificity in a temporal manner by variable day-night expression. In parallel, age-related brain dysfunction and neurodegeneration are associated with abnormal intracellular concentrations of calcium. Here, we investigated whether daily changes in the number of CR neurons are a feature of the aged IGL and vLGN in rats. To this end, we perfused rats, ranging from 3 to 24 months of age, within distinct phases of the day, namely zeitgeber times (ZTs). Then, we evaluated CR immunolabeling through design-based stereological cell estimation. We observed distinct daily rhythms of CR expression in the IGL and in both the retinorecipient (vLGNe) and non-retinorecipient (vLGNi) portions of the vLGN. In the ZT 6, the middle of the light phase, the CR cells are reduced with aging in the IGL and vLGNe. In the ZT 12, the transition between light to dark, an age-related CR loss was found in all nuclei. While CR expression predominates in specific spatial domains of vLGN, age-related changes appear not to be restricted at particular portions. No alterations were found in the dark/light transition or in the middle of the dark phase, ZTs 0, and 18, respectively. These results are relevant in the understanding of how aging shifts the phenotype of visual related cells at topographically organized channels of visuomotor and circadian processing.

Keywords: aging; calcium binding proteins; calretinin; circadian rhythms; intergeniculate leaflet; lateral geniculate body; stereology; ventral lateral geniculate nucleus.

Figure 1

Delineation of IGL, vLGNe, and vLGNe in CR immunolabeled sections at distinct rostrocaudal positions. Distance caudal (–) to Bregma 0.0 (in mm) are indicated at the top of each panel. In the rat lateral geniculate body, dLGN, IGL, and vLGN are bordered laterally by the opt. (A) At −4.2 mm, vLGNi weak background aids in the delineation of its boundary with vLGNe. (B,C) At −4.5 and −4.8 mm, the medial portion of IGL projects ventrally and stronger immunoreactivity in CR cell bodies denoting the vLGNe/vLGNi border. Scale bar = 200 μm.

Figure 2

Photomicrographs of CR immunolabeled histological sections containing the IGL, vLGNe, and vLGNi in animals from distinct age and ZT groups. CR sections are obtained after brain perfusion at ZTs 0 (dark/light phase), 6 (light phase), 12 (light/dark phase), and 18 (dark phase) from young (A–D), middle-aged (E–H), or old (I–L) animals. Scale bar = 225 μm.

Figure 3

High-magnification photomicrographs of CR immunolabeled cells of the IGL, vLGNe, and vLGNi at ZT 12. The IGL, vLGNe, and vLGNiCR+ neurons from young (A–C), middle-aged (D–F), and old (G–I) animals are shown in detail. Scale bar = 50 μm.

Figure 4

Daily rhythmicity of CR expression in the IGL, vLGNe, and vLGNi. Stereological estimations of total CR cell number at each ZT in the IGL (A), vLGNe (B), and vLGNi (C) from young (black), middle-aged (red), or old (blue) animals. Data from ZT 0 are double plotted as ZT 24, to graphically represent the 24 h of the day. Shaded gray areas represent the dark phase. Blue *p < 0.05 in young vs. old comparison. Blue #p < 0.05 in middle-aged vs. old comparison. Red *p < 0.05 in young vs. old comparison. Data are analyzed by two-way ANOVA followed by Tukey's test for post-hoc comparison. Data are plotted as mean ± SD.

Figure 5

Age-related changes in the CR expression of the IGL, vLGNe, and vLGNi. Total CR+ cell numbers of the IGL (A–D), vLGNe (E–H), and vLGNi (I–L) are plotted as a function of age in each ZT. Pearson's correlation coefficient (r), regression lines with 95% confidence intervals and p-values are shown when a significant linear relation is observed.

Figure 6

Spatial distribution of CR staining in the vLGN. (A) Photomicrograph of the vLGN digitally processed for background subtraction. The vLGN borders are drawn (yellow) and equally spaced lines (gray) in the lateral to medial axis are overlaid to plot profiles of pixel intensity variations in arbitrary units (AU). Scale bar = 100 μm. (B–I) CR staining levels through the lateral to medial axis in the young and old animals at t ZTs 0 (blue), 6 (red), 12 (green), and 18 (black). (J,K) Every ZT plot overlaid for the young and old animals. Relative CR predominance distinguishes five zones, namely, e1, e2, e3, e4, and vLGNi domains. Data are plotted as mean gray values with standard error for each group.