Wholemount immunohistochemistry of the cerebellum reveals striped Purkinje cell loss across the cerebellar cortex of some aged mice.

A) Wholemount cerebellum of a 16-month-old mouse immunostained for calbindin and viewed from different angles. D = dorsal; L = lateral; V = ventral; A = anterior; P = posterior. Scale bar = 2 mm. B) High-magnification images of Purkinje cells in the wholemount cerebellum of an aged mouse. Dotted lines indicate stripes of surviving Purkinje cells, and the white arrowhead indicates an axonal torpedo. C) Schematic of the pattern of age-related Purkinje cell loss based on wholemount cerebella, where dark gray stripes represent bands largely composed of surviving Purkinje cells and white stripes represent bands where most Purkinje cells have degenerated. Cerebellar lobules are labeled with Roman numerals. D-I) Wholemount cerebella of mice immuno-stained for calbindin and viewed from the anterior zone: D) 2-month-old mouse; E) 15-month-old mouse without Purkinje cell loss; F) 23-month-old mouse without Purkinje cell loss; G) 14-month-old mouse with striped Purkinje cell loss; H) 15-month-old mouse with striped Purkinje cell loss; I) 16-month-old mouse with striped Purkinje cell loss. Asterisks indicate staining artifacts, either caused by continuous rubbing of the cerebellum during staining (hemispheres in panels D-F and lobules I-II in panel G) or accidental removal of surface tissue during dissection (lobules I-II in panel F). Arrows indicate bands of surviving Purkinje cells that are consistent across mice with striped Purkinje cell loss. D = dorsal; L = lateral; V = ventral. Cerebellar lobules are labeled with Roman numerals.

Information from mice used in this study.

Mice from the same litter are indicated with the same letter.

Aged mice with striped Purkinje cell loss show cellular level anatomical hallmarks of degeneration.

A) Coronal cut cerebellar tissue sections of lobule III immunostained for calbindin. Black arrowheads indicate thickened axons, and pink arrowheads indicate axonal torpedoes. Dashed lines indicate the Purkinje cell layer (PCL). Scale bar = 100 μm. B) Coronal cut cerebellar tissue sections of lobule VIII immunostained for calbindin. Asterisks indicate shrunken dendritic arbors. Scale bar = 100 μm. C) Quantification of molecular layer thickness in lobule VIII; **** indicates p ≤ 0.0001. D) Coronal cut cerebellar tissue sections either stained with Neutral Red or immunostained for calbindin and stained with Neutral Red. Asterisks indicate Purkinje cell bodies. Scale bar = 100 μm; inset scale bar = 50 μm.

Cerebella of aged Purkinje cell-specific fluorescent reporter mice display the same pattern of Purkinje cell loss as revealed by wholemount calbindin immunohistochemistry.

A) Wholemount cerebella of Purkinje cell-specific fluorescent reporter mice visualized with blue light and viewed from different angles. Cerebellar lobules are labeled with Roman numerals. D = dorsal; L = lateral; V = ventral; A = anterior; P = posterior. B) Schematics of the dorsal view of cerebella from young, middle-aged, and older Purkinje cell-specific fluorescent reporter mice. Lighter colors indicate less intense reporter expression. C) Schematics of sagittal sections of the cerebellum and a wholemount cerebellum indicating the location of tissue sections. Dashed lines indicate the position and angle of tissue sections. D) Coronal cut cerebellar tissue sections of Purkinje cell-specific fluorescent reporter mice immunostained for calbindin and GFP. Dashed lines indicate boundaries between surviving Purkinje cells and degenerating Purkinje cells. Scale bar = 250 μm; inset scale bar = 100 μm.

The pattern of age-related Purkinje cell loss has some similarities to zebrin II expression, but the unique overall map represents a greater cerebellar complexity.

A) Schematics of wholemount cerebella and sagittal sections of the cerebellum indicating the location of tissue sections. Green indicates calbindin expression and alternating green and magenta indicates where calbindin and zebrin II are co-expressed. Dashed lines indicate the position and angle of tissue sections. Cerebellar lobules are labeled with Roman numerals. B) Coronal cut tissue sections co-stained for calbindin (green) and zebrin II (magenta). Zebrin II-positive stripes are indicated by P1, P2, and P3. Brackets indicate bands of degenerating Purkinje cells, and asterisks indicate bands of degenerating Purkinje cells in uniformly zebrin II-positive regions. Scale bar = 250 μm. C) Schematics of half of a wholemount cerebellum indicating calbindin and zebrin II expression (alternating green and magenta) and bands of surviving Purkinje cells as indicated by calbindin expression (dark gray). D) Paraflocculi of wholemount cerebella immunostained for calbindin and viewed from different angles. Cerebellar lobules are labeled with Roman numerals. D = dorsal; L = lateral; M = medial; V = ventral; A = anterior; P = posterior.

Regions with lasting resistance to Purkinje cell loss during normal aging are revealed in serial sections from 25-month-old mouse.

Coronal cut cerebellar tissue sections immunostained for calbindin and arranged in order from anterior to posterior. Cerebellar lobules are labeled with Roman numerals. D = dorsal; L = lateral; V = ventral; A = anterior; P = posterior. Scale bar = 250 μm.

Regions with apparent resistance to cell loss in 25-month-old mouse have Purkinje cells with extreme morphological abnormalities.

High-magnification images of cerebellar tissue sections immunostained for calbindin. Asterisks indicate recurrent axon collaterals, and arrows indicate thickened dendrites. Scale bar = 50 μm.

Aged mice display impaired performance on the accelerating rotarod and increased tremor but no deficits on the horizontal ladder.

A) Schematics of motor function tests. B) Latency to fall from accelerating rotarod. Error bars indicate standard error of the mean. B’) Latency to fall with aged mice sorted based on the presence or absence of Purkinje cell loss. C) Number of footslips when crossing the horizontal ladder. ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, and **** indicates p ≤ 0.0001. C’) Number of footslips with aged mice sorted based on the presence or absence of Purkinje cell loss. D) Power spectrum of tremor detected by a tremor monitor. Error bars indicate standard error of the mean. D’) Power spectrum of tremor with aged mice sorted based on the presence or absence of Purkinje cell loss.

Aging humans have Purkinje cell degeneration that can be visualized with calbindin.

Asterisks indicate the remaining Purkinje cell bodies. Dashed lines indicate the boundaries of Purkinje cell dendritic arbors. Scale bar = 100 μm.

The presence of Purkinje cell loss varies across aged mice.

A) Proportion of aged mice with striped Purkinje cell loss, non-striped Purkinje cell loss, and no Purkinje cell loss divided by sex. B) Mediolateral inverse pixel intensity of Purkinje cell labeling in aged mice. Inverse pixel intensity was calculated within identically sized rectangular regions of interest placed over lobules II and III in coronal cut tissue sections immuostained for calbindin. Data were grouped according to whether mice had non-striped Purkinje cell loss (blue) or striped Purkinje cell loss (black). B’) Mediolateral inverse pixel intensity of Purkinje cell labeling separated by individual aged mouse.

Different calbindin antibodies reveal matching expression patterns, including age-related striped Purkinje cell loss.

Coronal tissue sections taken from the anterior and posterior cerebellum of young and aged mice and co-immunostained with two different calbindin antibodies. Scale bar = 500 μm.

Calbindin and GFP reveal overlapping patterns of expression in aged mice with striped Purkinje cell loss.

A) Mediolateral pixel intensity of Purkinje cell labeling in aged mice. Pixel intensity was calculated within identically sized rectangular regions of interest placed over lobules II and III in coronal cut tissue sections taken from aged Purkinje cell-specific fluorescent reporter mice and co-immunostained for calbindin and GFP. Calbindin pixel intensity is shown in magenta, and GFP pixel intensity is shown in green. A’) Mediolateral pixel intensity of Purkinje cell labeling separated by individual aged mice.

Patterned calbindin expression and staining artifacts can obscure Purkinje cell loss due to neurodegeneration.

A) Coronal cut tissue sections from C57BL/6J mice either immunostained for calbindin alone or immunostained for calbindin and stained with Neutral Red. Brackets indicate regions of zonal calbindin expression. Calbindin zones, when present, arise due to domains of high versus low expression rather than extended areas that completely lack calbindin-expressing Purkinje cells. Dashed lines indicate boundaries formed by intact Purkinje cell dendrites and degenerating dendrites. Arrowheads indicate calbindin-negative Purkinje cell bodies that are stained with Neutral Red. Cerebellar lobules are labeled with Roman numerals. Scale bar = 250 μm; inset scale bar = 100 μm. B) Coronal cut tissue sections from aged C57BL/6J mice either immunostained for calbindin alone or immunostained for calbindin and stained with Neutral Red. Arrowheads indicate Purkinje cell bodies that were not stained with calbindin antibody. Scale bar = 100 μm; inset scale bar = 50 μm.

Calbindin and Purkinje cell-specific fluorescent reporter expression reflect the same stripes of Purkinje cell loss with respect to zebrin II.

Coronal cut tissue sections taken from lobules II-III of young and aged Purkinje cell-specific fluorescent reporter mice and co-immunostained for GFP, zebrin II, and calbindin. Scale bar = 250 μm.

There is no correlation between weight, peak tremor power, or relative age.

A) Peak tremor power plotted against body weight, with point color indicating age and point size indicating sex. B) Correlation matrix of peak tremor power, body weight, and age calculated using Spearman’s correlation.