Martha L. Zettel

Effects of aging and sensory loss on glial cells in mouse visual and auditory cortices

Normal aging is often accompanied by a progressive loss of receptor sensitivity in hearing and vision, whose consequences on cellular function in cortical sensory areas have remained largely unknown. By examining the primary auditory (A1) and visual (V1) cortices in two inbred strains of mice undergoing either age‐related loss of audition (C57BL/6J) or vision (CBA/CaJ), we were able to describe cellular and subcellular changes that were associated with normal aging (occurring in A1 and V1 of both strains) or specifically with age‐related sensory loss (only in A1 of C57BL/6J or V1 of CBA/CaJ), using immunocytochemical electron microscopy and light microscopy. While the changes were subtle in neurons, glial cells and especially microglia were transformed in aged animals. Microglia became more numerous and irregularly distributed, displayed more variable cell body and process morphologies, occupied smaller territories, and accumulated phagocytic inclusions that often displayed ultrastructural features of synaptic elements. Additionally, evidence of myelination defects were observed, and aged oligodendrocytes became more numerous and were more often encountered in contiguous pairs. Most of these effects were profoundly exacerbated by age‐related sensory loss. Together, our results suggest that the age‐related alteration of glial cells in sensory cortical areas can be accelerated by activity‐driven central mechanisms that result from an age‐related loss of peripheral sensitivity. In light of our observations, these age‐related changes in sensory function should be considered when investigating cellular, cortical, and behavioral functions throughout the lifespan in these commonly used C57BL/6J and CBA/CaJ mouse models.

Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma.

We utilize the polarization and directionality of light emitted by fibrillar collagen via second harmonic generation to determine structural relationships between collagen in mouse mammary tumor models and the healthy mammary fat pad. In spite of the aberrations in collagen production and degradation that are the hallmarks of tumor stroma, we find that the characteristic angle of SHG scatterers within collagen fibrils, and the spatial extent over which they are appropriately ordered for SHG production, are the same in tumor and healthy collagen. This suggests that the SHG-producing subpopulation of collagen is unaffected by the altered collagen synthesis of the tumor stroma, and protected from its aberrant degradative environment.

Age-related changes in calbindin D-28k and calretinin immunoreactivity in the inferior colliculus of CBA/CaJ and C57Bl/6 mice

This study examines calbindin D‐28k and calretinin immunoreactivity in the inferior colliculus (IC) of young and old mice of two strains. The CBA/CaJ mouse maintains good hearing until very late in life, whereas the C57Bl/6 strain exhibits severe sensorineural hearing loss at an early age. Young and old mice of both strains were selected with matching auditory brainstem response audiograms and gap detection thresholds. Brain sections were reacted with anti‐calbindin D‐28k (CB) and anti‐calretinin (CR). Staining patterns were characterized and cell counts performed. CB immunoreactivity was high only in the nucleus of the commissure (NCO); counts revealed a 22.3% decrease in the number of CB+ cells in old CBA mice and a 25.1% decrease in old C57 mice. Calretinin immunoreactivity was high in the pericentral regions of the IC, but the central nucleus was devoid of CR+ cells. The dorsal cortex, lateral nucleus, and NCO showed increases of 42.3, 49.0, and 61%, respectively, in the number of CR+ cells, but only in the old CBA mice. No significant change was observed in the old C57 mice. Whereas decreases in CB immunoreactivity are common with age, this study is the first to report an age‐related increase in CR immunoreactivity in the auditory system. The increase in CR+ cells is a possible compensatory adaptation to the decrease in CB+ cells. That the number of CR+ cells remains constant with age in C57 mice suggests this compensation may depend upon stimulus‐driven activity, but this requires further study. J. Comp. Neurol. 386:92–110, 1997.

Calbindin D-28k immunoreactivity in the medial nucleus of the trapezoid body declines with age in C57BL/6, but not CBA/CaJ, mice.

This study compared calbindin D-28k immunoreactivity in the medial nucleus of the trapezoid body (MNTB) in young (3-4 month old) and old (24-26 month old) CBA/CaJ mice, and young (3-4 month old), middle-aged (6.5-8.5 month old), and old (24-29 month old) C57BL/6 mice. C57BL/6 mice exhibit progressively more severe peripheral (sensorineural) hearing loss between 4 and 12 months of age, whereas CBA/CaJ mice show little change in peripheral sensitivity until very late in life. We obtained auditory brainstem response audiograms on all subject mice. Old CBA mice were selected for study whose audiograms matched those of young CBA and C57 controls. Middle-aged C57 mice showed elevated thresholds indicative of peripheral degeneration. Brain sections were reacted with anti-calbindin D-28k (CB). Staining patterns in Nissl and anti-CB material were characterized and cells were counted. We found no significant change in the number of CB+ cells or the total number of cells in the MNTB of old CBA mice compared to young controls. However, the mean number of CB+ cells decreased by 11% in middle-aged, and by 14.8% in old C57 mice. Since the decline in C57 mice was significant by 6.5-8.5 months of age, the decrease could be the consequence of a loss of input from the cochlear nucleus where cell numbers are known to decline by this age in this strain. The total number of neurons in MNTB assessed from Nissl material showed a more modest 7.1% decline with age in C57 mice, implying that the greater loss of CB immunoreactive cells with age cannot be completely attributed to a reduction in the total number of cells.

Early bilateral deafening prevents calretinin up-regulation in the dorsal cortex of the inferior colliculus of aged CBA/CaJ mice

This study was conducted to test the hypothesis that age-related calretinin (CR) up-regulation seen in the dorsal cortex of the inferior colliculus (ICdc) of old hearing CBA mice is dependent upon neural activity within the auditory pathway. We tested this hypothesis by bilaterally deafening young CBA/CaJ mice with kanamycin, and then aging them until 24 months. This manipulation mimics the lack of sound-evoked auditory activity experienced by old C57BL/6J mice, who are deaf and do not show CR up-regulation with age. Cell counts revealed that the density of CR+ cells in the ICdc of old hearing CBA mice was statistically different from old deafened CBA mice raised under identical conditions. Old hearing CBAs possessed an average of 27.54 more CR+ cells/100 microm2 than old deafened CBAs. When old deafened CBAs were compared to young hearing CBAs, young hearing C57s, and old deaf C57s, there was no significant difference in mean CR+ cell density in ICdc. Thus, only the old normal hearing CBAs showed an increase in CR+ cells with age, supporting the hypothesis that CR up-regulation depends upon sound-evoked activity. Moreover, these results demonstrate that up-regulation of CR expression was not simply due to a mouse strain difference.

Glutamate-related gene expression changes with age in the mouse auditory midbrain.

Glutamate is the main excitatory neurotransmitter in both the peripheral and central auditory systems. Changes of glutamate and glutamate-related genes with age may be an important factor in the pathogenesis of age-related hearing loss-presbycusis. In this study, changes in glutamate-related mRNA gene expression in the CBA mouse inferior colliculus with age and hearing loss were examined and correlations were sought between these changes and functional hearing measures, such as the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs). Gene expression of 68 glutamate-related genes was investigated using both genechip microarray and real-time PCR (qPCR) molecular techniques for four different age/hearing loss CBA mouse subject groups. Two genes showed consistent differences between groups for both the genechip and qPCR. Pyrroline-5-carboxylate synthetase enzyme (Pycs) showed down-regulation with age and a high-affinity glutamate transporter (Slc1a3) showed up-regulation with age and hearing loss. Since Pycs plays a role in converting glutamate to proline, its deficiency in old age may lead to both glutamate increases and proline deficiencies in the auditory midbrain, playing a role in the subsequent inducement of glutamate toxicity and loss of proline neuroprotective effects. The up-regulation of Slc1a3 gene expression may reflect a cellular compensatory mechanism to protect against age-related glutamate or calcium excitoxicity.

Serotonin 2B receptor: Upregulated with age and hearing loss in mouse auditory system

Serotonin (5-HT) is a monoamine neurotransmitter. Serotonin may modulate afferent fiber discharges in the cochlea, inferior colliculus (IC) and auditory cortex. Specific functions of serotonin are exerted upon its interaction with specific receptors; one of those receptors is the serotonin 2B receptor. The aim of this study was to investigate the differences in gene expression of serotonin 2B receptors with age in cochlea and IC, and the possible correlation between gene expression and functional hearing measurements in CBA/CaJ mice. Immunohistochemical examinations of protein expression of IC in mice of different age groups were also performed. Gene expression results showed that serotonin 2B receptor gene was upregulated with age in both cochlea and IC. A significant correlation between gene expression and functional hearing results was established. Immunohistochemical protein expression studies of IC showed more serotonin 2B receptor cells in old mice relative to young adult mice, particularly in the external nucleus. We conclude that serotonin 2B receptors may play a role in the pathogenesis of age-related hearing loss.

Distribution of calbindin D-28k immunoreactivity in the cochlear nucleus of the young adult chinchilla

Calbindin is a 28 kD calcium-binding protein found in neural tissue. Although its functional role in nerve cell physiological processing is still uncertain, previous investigations have suggested that because of its intracellular calcium buffering and regulation properties, it could influence temporal precision of neuronal firing to subserve temporal processing in the auditory brainstem, or could mediate monaural versus binaural coding, or be involved in synaptic plasticity (learning). The present study demonstrates differential calbindin immunoreactivity in the cochlear nuclear complex of the chinchilla, a rodent with exceptionally good low-frequency hearing. The most intense labeling in the cochlear cochlear nucleus was in somata of cartwheel and fusiform cells of the fusiform cell layer, and somata and process of the molecular layer of the dorsal cochlear nucleus (DCN). Only a relatively few scattered neurons were stained in the deep layers of DCN. In contrast, moderate labeling of neurons and neuropil throughout the ventral cochlear nucleus was seen. For instance, moderately stained spherical and elongate cells of the anteroventral cochlear nucleus were observed in contact with labeled puncta and amidst stained fibers. In the cochlear nerve root region, stained auditory nerve fibers and global cells were noted. In the posteroventral cochlear nucleus, principal cells of elongate and octopus shape were observed, in contact with labeled swellings and surrounded by labeled neuropil.

The effects of early bilateral deafening on calretinin expression in the dorsal cochlear nucleus of aged CBA/CaJ mice.

The aim of this study was to test the hypothesis that calretinin (CR) levels in the aged mouse auditory brainstem depend upon hearing ability. Old animals with good hearing, and thus higher sound-evoked activity levels, were predicted to have higher levels of CR immunoreactivity than old animals with hearing loss. CR immunoreactivity was analyzed in the deep layer (layer III) of the dorsal cochlear nucleus (DCN) in CBA/CaJ mice that were bilaterally deafened at 3 months of age with kanamycin, and then aged until 24 months. This manipulation partially mimics the lack of sound-evoked auditory activity experienced by old C57BL/6J mice, who are deaf at 24 months of age (but show residual hearing at 15 months) and have lower levels of CR immunoreactivity than old CBA mice with normal hearing [Hear. Res. 158 (2001) 131]. Cell counts revealed that the density of CR+ cells in DCN layer III of the deafened CBA mice was statistically different from old intact CBA mice raised under identical conditions. Old deafened CBAs showed a decline of 47% in the mean density of CR+ cells compared to old hearing CBAs, thus supporting the hypothesis. Interestingly, while there tended to be fewer CR+ cells in the old deaf C57s as compared to young C57s and young and old CBAs with normal hearing, the difference was not statistically significant. It is possible that the residual hearing of C57 mice at 15 months may provide sufficient auditory input to maintain CR at levels higher than CBA mice that are deafened completely at 3 months of age, and are profoundly deaf for a much longer time (21 months).

Tumor-associated macrophages and stromal TNF-α regulate collagen structure in a breast tumor model as visualized by second harmonic generation

Abstract. Collagen fibers can be imaged with second harmonic generation (SHG) and are associated with efficient tumor cell locomotion. Preferential locomotion along these fibers correlates with a more aggressively metastatic phenotype, and changes in SHG emission properties accompany changes in metastatic outcome. We therefore attempted to elucidate the cellular and molecular machinery that influences SHG in order to understand how the microstructure of tumor collagen fibers is regulated. By quantifying SHG and immunofluorescence (IF) from tumors grown in mice with and without stromal tumor necrosis factor (TNF)-α and in the presence or absence of tumor-associated macrophages (TAMs), we determined that depletion of TAMs alters tumor collagen fibrillar microstructure as quantified by SHG and IF. Furthermore, we determined that abrogation of TNF-α expression by tumor stromal cells also alters fibrillar microstructure and that subsequent depletion of TAMs has no further effect. In each case, metastatic burden correlated with optical readouts of collagen microstructure. Our results implicate TAMs and stromal TNF-α as regulators of breast tumor collagen microstructure and suggest that this regulation plays a role in tumor metastasis. Furthermore, these results indicate that quantification of SHG represents a useful strategy for evaluating the cells and molecular pathways responsible for manipulating fibrillar collagen in breast tumor models.