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, and 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 show anatomical hallmarks of Purkinje cell degeneration.

A) Coronal 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 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 ≤ 0.001. D) Coronal 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 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 the zebrin II expression, but their unique overall maps represent 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 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 cerebellar tissue sections immunostained for calbindin and arrayed 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.

Degeneration-resistant regions 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 presence or absence of Purkinje cell loss. C) Number of footslips when crossing 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 presence or absence of Purkinje cell loss. D) Power spectrum of tremor detected by tremor monitor. Error bars indicate standard error of the mean. D’) Power spectrum of tremor with aged mice sorted based on presence or absence of Purkinje cell loss.

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

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

The presence of Purkinje cell loss varies across aged mice.

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

Scale bar = 500 μm.

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

A) Coronal 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. 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 tissue sections from aged C57BL/6J mouse 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.

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

P-values were calculated using Spearman’s correlation.